METHOD OF PERFORMING HIGH DENSITY MULTIPLEX STAINING

FOR IN SITU HYBRIDIZATION

CROSS REFERENCE TO RELATED APPLICATION This application claims the benefit of the earlier filing date of U.S.

Provisional Application No. 61/070,293, filed March 21, 2008, U.S. Provisional Application No. 61/070,439, filed March 21, 2008, and U.S. Provisional Application No. 61/070,328, filed March 21, 2008, all of which are incorporated herein by reference.

FIELD

The disclosure relates to methods of practicing immunohistochemistry, and in situ hybridization analysis.

BACKGROUND

Immunohistochemistry (IHC) is a technique involving the use of specific binding agents, such as antibodies and antibody fragments, to detect specific antigens that may be present in a tissue sample. Immunohistochemistry is widely used in clinical and diagnostic applications, for example to diagnose particular disease states or conditions, such as a cancer. For example, a diagnosis of a particular type of cancer can be made based on the presence of a particular marker antigen present in a sample obtained from a subject. Immunohistochemistry can utilize various detectable molecules that can be associated with the specific binding agents, such as, fluorescent, chromogenic, and chemiluminescent molecules. Biological samples can also be examined using in situ hybridization techniques, such as Chromogenic In Situ Hybridization (CISH) and Fluorescence In Situ Hybridization (FISH), collectively referred to as ISH. Each method has its advantages and disadvantages. Currently ISH users select either CISH or FISH based on personal preference, the particular. application, the instructions in a reagent kit or the type of microscope available. Because of this selection process, the advantages of the method not chosen are lost.

Immunohistochemical and ISH methods that are used to capture information are becoming increasing important in research and clinical settings. Improvements to these methods are needed to increase accuracy and the relative ease of practicing such methods.

SUMMARY

Disclosed herein are embodiments of a method for analyzing samples that use multiplexed reactions with targeting moieties that are peptide-based. Particular examples include contacting a sample with a first set of probes that include more than one probe and some examples further capture a first image to detect the first set of probes. The first set of probes are then substantially released from the sample, for example by heating the sample at a temperature of about 50° C to about 100° C for a period of time of about 5 to about 30 minutes. A second set of probes that include more than one probe can be added to the sample and the signal from the second set of probes can be detected. Thus, information relating to multiple targets can be collected from a single sample.

In some examples, more than 2, 3, 4, or 5 different probes can be used to detect multiple targets. In some examples, the sample is maintained at the desired temperature for greater than 5, 10, 15, 20, 25, 30, 40, 50, or 60 minutes. In some examples, the sample is heated at no more than 110 ⁰ C, for example from about 60°C to about 90 ⁰ C. One of ordinary skill in the art will appreciate that the probes can be released from the sample and the sample can be re-probed multiple times. Additionally, images, such as digital images, can be captured of the sample with the various probes sets bound. These images can be manipulated, for instance by overlaying them to consolidate the information to a single image.

The methods provided herein can be used to determine if a subject from whom the sample was obtained has a particular disorder, and can be used to detect multiple targets in tissue samples. For example, the methods can be used to identify at least about 15, 20, 25, 30, or at least about 35 targets in a single sample. The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating one embodiment of a method for amplifying detection signals using haptens coupled to primary antibodies and anti- hapten antibodies coupled to enzymes useful for reacting with labels.

FIGS. 2A-2B are images of a tonsil stained with kappa, lambda, and cytokeratin in three different cycles.

FIGS. 3 A-3C are images illustrating using combinations of kappa and lambda. FIG. 3 A shows a combination of kappa and lambda, FIG. 3B shows a combination of kappa, lambda, and cytokeratin, and FIG. 3C shows FIG. 3B converted to a brightfield image.

FIGS. 4 A-4C are images of adenocarcinoma stained with probe to mRNA by FISH. FIG. 4A is a FISH image as a red fluorescent signal. FIGS. 4B and 4C are images of the same tissue after heat recycling. FIG. 4B is shown with increased brightness to reveal some of the microscopic detail, and FIG. 4C is shown at the same brightness level as FIG. 4A. These results show nearly complete stripping of the original ISH stain.

FIG. 5 is an image of adencarcinoma stained with a probe to mRNA by FISH. FIG. 6 is an image of adencarcinoma stained with a counterstain and viewed by brightfield microscopy.

FIG. 7 is an overlay of FIGS. 4A, 5, and 6 to form an assembled multiplex image.

DETAILED DESCRIPTION

I. Terms

Unless otherwise noted, technical terms are used according to conventional usage. As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B. It is further to be understood that all

temperatures and times provided herein are approximate. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various examples In order to facilitate review of the various examples of this disclosure, the following explanations of specific terms are provided:

Analyzing: A process of assimilating information. For example, detecting through the use of a sensory organ or a computer aided device specific features of sample. Exemplary, non-limiting examples of features include probe binding and cellular structures. In some examples, analyzing includes comparing the detected features to a standard, control, or reference data set.

Antibody: A polypeptide ligand that includes at least a light chain or heavy chain immunoglobulin variable region and specifically binds an epitope of an antigen. Antibodies can include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, or fragments of antibodies, and the like. In some examples, an antibody-based targeting moiety can be directly, or indirectly, associated with a label.

The term "specifically binds" refers to, with respect to an antibody based targeting moiety, the preferential association of an antibody to a target molecule. Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of a targeting moiety, such as a bound antibody or other ligand (per unit time), to a target polypeptide, such as compared to a non-target polypeptide. A variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular target molecule. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory

Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreacti vity .

Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab ¹ fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies) and heterocoηjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

A "monoclonal antibody" is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies. Antigen: A molecule, or portion of a molecule, that can stimulate an immune response, for example in a mammal. Antigens are usually proteins or polysaccharides. An epitope is an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response. An antibody binds a particular antigenic epitope. The binding of an antibody to a particular antigen or epitope of an antigen can be used to localize the position of the antigen, for example in or on a biological sample, or to determine if the particular antigen is present in a biological sample.

Avidin: Any type of protein that specifically binds biotin to the substantial exclusion of other small molecules that might be present in a biological sample. Examples of avidin include avidins that are naturally present in egg white, oilseed protein (e.g., soybean meal), and grain (e.g., corn/maize) and streptavidin, which is a protein of bacterial origin.

Binding affinity: The tendency of one molecule to bind (typically non- covalently) with another molecule, such as the tendency of a targeting moiety to bind to a target. Binding affinity can be measured as a binding constant. A target molecule and a targeting moiety (such as an antibody/antigen pair or nucleic acid probe/nucleic acid sequence pair) can display a binding constant such as at least 1 x 10 ⁵ M ^"1 , such as at least 1 x 10 ⁶ M ^"1 , at least 1 x 10 ⁷ M ^'1 or at least 1 x 10 ⁸ M ^"1 . In one embodiment, binding affinity is calculated by a modification of the Scatchard method described by Frankel et al., MoI. Immunol, 16: 101-106, 1979. In another example, binding affinity is measured by the melting temperature (i.e., disassociation temperature) of a double stranded nucleic acid sequence, such as a targeting moiety bound to a target nucleic acid molecule. In other examples, binding affinity is measured by an antigen/antibody dissociation rate. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay. In several examples, a high binding affinity for an antibody/antigen pair is at least about 1 x 10 ⁸ M ^"1 . In other embodiments, a high binding affinity is at least about 1.5 x 10 ⁸ M ^"1 , at least about 2.0 x 10 ⁸ M ^"1 , at least about 2.5 x 10 ⁸ M ^'1 , at least about 3.0 x 10 ⁸ M ^'1 , at least about 3.5 x 10 ⁸ M ^"1 , at least about 4.0 x 10 ⁸ M ^'1 , at least about 4.5 x 10 ⁸ M ^"1 , or at least about 5.0 x 10 ⁸ M ^"1 .

Chimeric antibody: An antibody that has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species.

Conditions sufficient to detect: Any environment that permits the desired activity, for example, that permits a probe to bind a target and the interaction to be detected. For example, such conditions include appropriate temperatures, buffer solutions, and detection means, such as microscopes and digital imaging equipment.

Conjugating, joining, bonding or linking: Covalently linking one molecule to another molecule to make a larger molecule. For example, making two

polypeptides into one contiguous polypeptide molecule, or to covalently attaching a hapten or other molecule to a polypeptide, such as an scFv antibody. In the specific context, the terms include reference to joining targeting moiety to a label. The linkage can be either by chemical or recombinant means. "Chemical means" refers to a reaction between the targeting moiety and the label such that there is a covalent bond formed between the two molecules to form one molecule.

Contacting: Refers to the relatively close physical proximity of one object to another object. Generally, contacting involves placing two or more objects in close physical proximity to each other to give the objects and opportunity to interact. For example, a sample can be contacted with a probe such that the probe has the opportunity to bind to a target.

Counterstain: Includes dyes or other detectable agents that can be used to detect cell structures (such as the nucleus, cytoskeleton, and the like). In some examples, counterstains are used to observe cells structures in samples that have been contacted with probes. Thus, counterstains allow the sample to be analyzed for both cell structures and the presence of the target molecule.

Coupled: When applied to a first atom or molecule being "coupled" to a second atom or molecule can be both directly coupled and indirectly coupled. A secondary antibody provides an example of indirect coupling. One specific example of indirect coupling is a rabbit anti-hapten primary antibody that is bound by a mouse anti-rabbit IgG antibody, which is in turn bound by a goat anti-mouse IgG antibody that is covalently linked to a label.

Data set: Refers to information obtained after a sample has been targeted with a probe. In instances where multiple probes are used, a data set can include information from multiple probes pertaining to multiple targets. In some examples a data set is captured as an image, such as a digital image.

Detecting: Refers to any method of determining if something exists, or does not exist, such as determining if a target molecule is present in a biological sample. For example, detecting includes using a sensory organ or a mechanical device to determine if a sample displays a specific characteristic. In certain examples, detection refers to visually observing a probe bound to a target, or observing the lack of binding of a probe to a target.

Disorder: A condition that is not the average condition displayed by a population. A disorder may or may not be indicative of a disease. Exemplary disorders include a tumor, infection from a microorganism, or genetic abnormality. Fluorescent tyramide derivative: Any molecule that includes a tyramide moiety that is capable of emitting a fluorescent signal when excited at an appropriate wavelength of light. Exemplary, non-limiting examples of such molecules include fluorescyl tyramide, tetramethyl-rhodamine tyramide, cyanine-3 -tyramide, and cy anine-5 -tyramide .

Hapten: A molecule, typically a small molecule that can combine specifically with an antibody, but typically is substantially incapable of being immunogenic except in combination with a carrier molecule.

Humanized immunoglobulin: An immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a "donor," and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences.

Imniunohistochemistry (IHC): A method of determining the presence or distribution of an antigen in a sample by detecting interaction of the antigen with an antibody. A sample including an antigen (such as a target antigen) is incubated with an antibody under conditions permitting antibody-antigen binding. Antibody- antigen binding can be detected by means of a detectable label conjugated to the antibody (direct detection) or by means of a detectable label conjugated to a secondary antibody, which is raised against the primary antibody (e.g., indirect detection). Detectable labels include, but are not limited to, radioactive isotopes, fluorochromes (such as fluorescein, fluorescein isothiocyanate, and rhodamine), and chromogenic molecules.

Label: Refers to a molecule that provides a detectable signal. Labels include molecules such as fluorescent molecules, chromogenic molecules and chemiluminescent molecules.

Linker peptide: A peptide that serves to link probe components together. For example, within an antibody binding fragment (such as an Fv fragment) a linker can serve to indirectly bond the variable heavy chain to the variable light chain. "Linker" can also refer to a peptide serving to link a targeting moiety, such as a scFv, to an enzymatic activity that interacts with a label, or to directly link a targeting moiety to a label. Mammal: This term includes both human and non-human mammals.

Similarly, the term "subject" includes both human and veterinary subjects.

Molecule of interest or Target: A pre-selected molecule, for example whose detection, location and/or concentration is to be determined. Non-limiting examples of targets include amino acid and nucleic acid sequences. Various other biomolecules can also be present with the target molecule. For example, the target molecule can be present in a biological sample (which can include other nucleic acid molecules and proteins).

Multiplex, -ed, -ing: Embodiments of the present disclosure allow multiple targets in a sample to be detected substantially simultaneously, or sequentially, as desired, using plural different probes. Multiplexing can include identifying and/or quantifying nucleic acids generally, DNA, RNA, peptides, proteins, both individually and in any and all combinations. Multiplexing also can include detecting two or more of a gene, a messenger RNA and a protein in a cell in its anatomic context. Neoplasia and Tumor: The process of abnormal and uncontrolled growth of cells. Neoplasia is one example of a proliferative disorder. In some examples, targets are used to determine whether a sample contains a neoplastic or tumor cells.

The product of neoplasia is a neoplasm (a tumor), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Examples of hematological tumors include leukemias, including acute leukemias (such as acute lymphocytic

leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocyte, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

Probe: As used herein a "probe" includes a targeting moiety capable of being directly or indirectly labeled. The probe can be directly conjugated to the label or it can be indirectly associated with the label. For example, a probe can include a targeting moiety that is conjugated to another molecule, such as biotin, and biotin can then be contacted with streptavidin conjugated to a label. Thus, the probe is indirectly associated with the label and all of these components are considered part of the probe. In another example, a probe is indirectly associated with a label in that the targeting moiety can include an enzymatic activity which when placed in contact with a substrate causes the substrate to be converted to a label which is deposited at the site of the target. Hence, the label is not conjugated to the targeting

moiety, but the combination of the targeting moiety and the label is considered a probe.

Quantum dot: A nanoscale particle that exhibits size-dependent electronic and optical properties due to quantum confinement. Quantum dots can be used as labels. Quantum dots have, for example, been constructed of semiconductor materials (e.g., cadmium selenide and lead sulfide) and from crystallites (grown via molecular beam epitaxy), etc. A variety of quantum dots having various surface chemistries and fluorescence characteristics are commercially available from Invitrogen Corporation, Eugene, Oregon (see, for example, U.S. Patent Nos. 6,815,064, 6,682596 and 6,649,138, each incorporated by reference herein).

Quantum dots are also commercially available from Evident Technologies (Troy, NY). Other quantum dots include alloy quantum dots such as ZnSSe, ZnSeTe, ZnSTe, CdSSe, CdSeTe, ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe, ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe, ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, and InGaN quantum dots (Alloy quantum dots and methods for making the same are disclosed, for example, in U.S. Application Publication No. 2005/0012182 and PCT Publication WO 2005/001889).

Sample: A sample, such as a biological sample, includes biological materials (such as nucleic acid molecules and proteins) obtained from an organism or a part thereof, such as a plant, animal, bacteria, and the like. In particular embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample includes any solid or fluid sample obtained from, excreted by, or secreted by any living organism, including without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas, among others, and multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer). A biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint

(for example, a normal joint or a joint affected by disease, such as a rheumatoid arthritis, osteoarthritis, gout or septic arthritis). A biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ. In some examples, a sample is a tissue or tumor biopsy.

Sequence identity: The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.

Methods for aligning sequences for comparison are well known in the art. These methods can be used to design targeting moieties that specifically target molecules in samples. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981 ; Needleman & Wunsch, J. MoI. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene 73:237-244, 1988; Higgins & Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Huang, et al., CABIOS 8:155-165, 1992; and Pearson et al., Methods in Molecular Biology 24:307-331, 1994. Altschul et al., J. MoI. Biol. 215:403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLASTTM; Altschul et al., J. MoI. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. BLASTTM can be accessed on the Internet at the NCBI website. As used herein, sequence identity is commonly determined with the BLASTTM software set to default parameters. For instance, blastn (version 2.0) software can be used to determine sequence identity between two nucleic acid sequences using default parameters (expect = 10, matrix = BLOSUM62, filter = DUST (Tatusov and Lipmann, in preparation as of December 1, 1999; and Hancock and Armstrong, Comput. Appl. Biosci. 10:67-70, 1994), gap existence cost = 1 1, per residue gap

cost = 1, and lambda ratio = 0.85). For comparison of two polypeptides, blastp (version 2.0) software can be used with default parameters (expect 10, filter = SEG (Wootton and Federhen, Computers in Chemistry 17:149-163, 1993), matrix = BLOSUM62, gap existence cost = 11, per residue gap cost = 1, lambda = 0.85). Signal: A detectable change or impulse in a physical property that provides information. In the context of the disclosed methods, examples include electromagnetic signals such as light, for example light of a particular quantity or wavelength.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals (such as laboratory or veterinary subjects).

Substantially release: The term "substantially release" as used herein refers the separation of one molecule from another molecule. For example, a target is substantially released from a probe to which it was bound, when at least 50% of the bound probe is capable of being removed without substantially damaging the sample. In other examples, at least 60%, 70%, 80%, 90%, or 95% of the probe can be released from the target and separated from the sample.

Targeting moiety: A molecule that specifically binds to a target. A targeting moiety and its target can be described as a specific binding pair. Specific binding pairs are pairs of molecules that bind each other to the substantial exclusion of binding to other molecules (for example, targeting moieties and targets can have a binding constant that is at least 10 ³ M ^'1 greater, 10 ⁴ M ^"1 greater or 10 ⁵ M ^'1 greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample). Particular examples of targeting moieties include specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A), antibodies, nucleic acids sequences, and protein- nucleic acids. In examples where the targeting moiety includes a protein it is referred to as a peptide-based probe.

II. Samples and Targets Samples include biological components and generally are suspected of including one or more target molecules of interest. Target molecules can be on the surface of cells and the cells can be in a suspension, or in a tissue section. Target

molecules can also be intracellular and detected upon cell lysis or penetration of the cell by a probe. One of ordinary skill in the art will appreciate that the method of detecting target molecules in a sample will vary depending upon the type of sample and probe being used. Methods of collecting and preparing samples are known in the art.

Samples used in the methods described herein, such as a tissue or other biological sample, can be prepared using any method known in the art. The samples can be derived from subjects that are suspected of having a disorder, such as a genetic abnormality or a neoplasia. The described methods can also be applied to normal samples. Such normal samples are useful, among other things, as controls for comparison to other samples. The samples can be analyzed for many different purposes. For example, the samples can be used in a scientific study or for the diagnosis of a suspected malady.

Samples can include multiple targets that can be specifically bound by a probe or probes. The targets can be nucleic acid sequences or peptides. Throughout this disclosure when reference is made to a target peptide it is understood that the nucleic acid sequences associated with that peptide can also be used as targets. In some examples, the target is a protein or nucleic acid molecule from a pathogen, such as a virus, bacteria, or intracellular parasite, such as from a viral genome. For example, a target protein may be produced from a target nucleic acid sequence associated with (e.g., correlated with, causally implicated in, etc.) a disease. In certain examples, the selected target protein is produced from a nucleic acid molecule associated with a neoplastic disease (or cancer). For example, the nucleic acid sequence can include at least one gene associated with cancer [e.g., HER2 (human epidermal growth factor receptor-2), c-Myc, n-Myc, AbI, Bcl2, Bcl6, RbI, p53, EGFR, TOP2A, MET, or genes encoding other receptors and/or signaling molecules, etc.] or chromosomal region associated with a cancer. The target protein can be produced from a nucleic acid sequence associated with a truncation that has been correlated with a cancer. A target nucleic acid sequence can vary substantially in size. Without limitation, the nucleic acid sequence can have a variable number of nucleic acid residues. For example a target nucleic acid sequence can have at least about 10

nucleic acid residues, or at least about 20, 30, 50, 100, 150, 500, 1000 residues. The probe can bind to the target nucleic acid sequence and provide a detectable signal.

Similarly, a target polypeptide can vary substantially in size. Without limitation, the target polypeptide will include at least one epitope that binds to the probe. In some embodiments that polypeptide can include at least two epitopes that bind to a probe. The probe can bind to the epitope and provide a detectable signal.

In specific non-limiting examples, a target protein is produced by a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) associated with a neoplasm (for example, a cancer). Numerous chromosome abnormalities (including translocations and other rearrangements, reduplication or deletion) have been identified in neoplastic cells, especially in cancer cells, such as B cell and T cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers and the like. Therefore, in some examples, at least a portion of the target molecule is produced by a nucleic acid sequence (e.g., genomic target nucleic acid sequence) reduplicated or deleted in at least a subset of cells in a sample.

Oncogenes are known to be responsible for several human malignancies. For example, chromosomal rearrangements involving the SYT gene located in the breakpoint region of chromosome 18ql 1.2 are common among synovial sarcoma soft tissue tumors. The t(18ql 1.2) translocation can be identified, for example, using probes with different labels: the first probe includes FPC nucleic acid molecules generated from a target nucleic acid sequence that extends distally from the SYT gene, and the second probe includes FPC nucleic acid generated from a target nucleic acid sequence that extends 3' or proximal to the SYT gene. When probes corresponding to these target nucleic acid sequences (e.g., genomic target nucleic acid sequences) are used in an in situ hybridization procedure, normal cells, which lack a t(18ql 1.2) in the SYT gene region, exhibit two fusion (generated by the two labels in close proximity) signals, reflecting the two intact copies of SYT. Abnormal cells with a t(l 8ql 1.2) exhibit a single fusion signal.

In other examples, a target protein produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is selected that is a tumor suppressor gene that is deleted (lost) in malignant cells. For example, the pl6 region (including D9S1749, D9S1747, pl6(INK4A), pi 4(ARF), D9S1748, pl5(INK4B), and

D9S1752) located on chromosome 9p21 is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the short arm of chromosome 1 (that encompasses, for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTLl, and SHGC-1322), and the pericentromeric region (e.g., 19pl3-19ql3) of chromosome 19 (that encompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCRl) ) are characteristic molecular features of certain types of solid tumors of the central nervous system.

The aforementioned examples are provided solely for purpose of illustration and are not intended to be limiting. Numerous other cytogenetic abnormalities that correlate with neoplastic transformation and/or growth are known to those of ordinary skill in the art. Target proteins that are produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences), which have been correlated with neoplastic transformation and which are useful in the disclosed methods, also include the EGFR gene (7pl2; e.g., GENBANK™ Accession No. NC_000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK™ Accession No. NC_000008, nucleotides 128817498-128822856), D5S271 (5pl5.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK™ Accession No. NC_000008, nucleotides 19841058-19869049), RBl (13ql4; e.g., GENBANK™ Accession No. NC_000013, nucleotides 47775912-47954023), p53 (17pl3.1 ; e.g., GENBANK™ Accession No. NC OOOO 17, complement, nucleotides 7512464-7531642)), N-MYC (2p24; e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides 151835231-151854620), CHOP (12ql3; e.g., GENBANK™ Accession No. NC OOOO 12, complement, nucleotides 56196638-56200567), FUS (16plL2; e.g., GENBANK™ Accession No. NC_000016, nucleotides 31098954-31110601), FKHR (13pl4; e.g., GENBANK™ Accession No. NC_000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK (2p23; e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides 29269144-29997936), Ig heavy chain, CCNDl (1 Iql3; e.g., GENBANK™ Accession No. NC OOOOl 1, nucleotides 69165054..69178423), BCL2 (18q21.3; e.g., GENBANK™ Accession No. NC OOOOl 8, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK™ Accession No. NC_000003, complement, nucleotides

188921859-188946169), MALFl, API (Ip32-p31 ; e.g., GENBANK™ Accession No. NC_000001, complement, nucleotides 59019051-59022373), TOP2A (17q21- q22; e.g., GENBANK™ Accession No. NC_000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No. NC_000021, complement, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK™ Accession No. NC_000021, complement, nucleotides 38675671-38955488); ETVl (7p21.3; e.g., GENBANK™ Accession No. NC_000007, complement, nucleotides 13897379-13995289), EWS (22ql2.2; e.g., GENBANK™ Accession No. NC_000022, nucleotides 27994271-28026505); FLIl (1 Iq24.1-q24.3; e.g., GENBANK™ Accession No. NCJ)OOOl 1 , nucleotides 128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides 222772851-222871944), PAX7 (Ip36.2- p36.12; e.g., GENBANK™ Accession No. NC OOOOOl, nucleotides 18830087-18935219, PTEN (10q23.3; e.g., GENBANK™ Accession No. NC_000010, nucleotides 89613175-89716382), AKT2 (19ql3.1-ql3.2; e.g., GENBANK™ Accession No. NCJ)OOO 19, complement, nucleotides 45431556-45483036), MYCLl (Ip34.2; e.g., GENBANK™ Accession No. NCJ)OOOOl, complement, nucleotides 40133685-40140274), REL (2pl3-pl2; e.g., GENBANK™ Accession No. NC_000002, nucleotides 60962256-61003682) and CSFlR (5q33-q35; e.g., GENBANK™ Accession No. NC 000005, complement, nucleotides 149413051-149473128).

In other examples, a target protein is selected from a virus or other microorganism associated with a disease or condition. Detection of the virus- or microorganism-derived target nucleic acid sequence {e.g., genomic target nucleic acid sequence) in a cell or tissue sample is indicative of the presence of the organism. For example, the target peptide, polypeptide or protein can be selected from the genome of an oncogenic or pathogenic virus, a bacterium or an intracellular parasite (such as Plasmodium falciparum and other Plasmodium species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, and Guardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species).

In some examples, the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from a viral genome.

Exemplary viruses and corresponding genomic sequences (GENBANK™ RefSeq Accession No. in parentheses) include human adenovirus A (NC_001460), human adenovirus B (NC_004001), human adenovirus C (NC OO 1405), human adenovirus D (NC 002067), human adenovirus E (NC_003266), human adenovirus F (NC OO 1454), human astrovirus (NC_001943), human BK polyomavirus (VO 1109; GI:60851) human bocavirus (NC 007455), human coronavirus 229E (NC_002645), human coronavirus HKUl (NC_006577), human coronavirus NL63 (NC 005831), human coronavirus OC43 ( NC_005147), human enterovirus A (NC_001612), human enterovirus B (NC_001472), human enterovirus C (NC_001428), human enterovirus D (NC_001430), human erythro virus V9 (NC _004295), human foamy virus (NC OO 1736), human herpesvirus 1 (Herpes simplex virus type 1) (NC OO 1806), human herpesvirus 2 (Herpes simplex virus type 2) (NC OO 1798), human herpesvirus 3 (Varicella zoster virus) (NCJ)01348), human herpesvirus 4 type 1 (Epstein-Barr virus type 1) (NC 007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC_009334), human herpesvirus 5 strain AD 169 (NC OOl 347), human herpesvirus 5 strain Merlin Strain (NC_006273), human herpesvirus 6A (NCJ)01664), human herpesvirus 6B (NC_000898), human herpesvirus 7 (NC OOl 716), human herpesvirus 8 type M (NC 003409), human herpesvirus 8 type P (NC 009333), human immunodeficiency virus 1 (NC OOl 802), human immunodeficiency virus 2 (NCJ)01722), human metapneumovirus

(NC 004148), human papillomavirus- 1 (NC OO 1356), human papillomavirus- 18 (NC 001357), human papillomavirus-2 (NC_001352), human papillomavirus-54 (NCJ)01676), human papillomavirus-61 (NC OO 1694), human papillomavirus-cand90 (NC 004104), human papillomavirus RTRX7 (NC 004761), human papillomavirus type 10 (NC_001576), human papillomavirus type 101 (NC 008189), human papillomavirus type 103 (NC 008188), human papillomavirus type 107 (NC 009239), human papillomavirus type 16 (NC OO 1526), human papillomavirus type 24 (NCJ)01683), human papillomavirus type 26 (NCJ)01583), human papillomavirus type 32 (NC OO 1586), human papillomavirus type 34 (NC OOl 587), human papillomavirus type 4 (NCJ)01457), human papillomavirus type 41 (NC OO 1354), human papillomavirus type 48 (NCJ)01690), human papillomavirus type 49 (NCJ)01591), human papillomavirus

type 5 (NC_001531), human papillomavirus type 50 (NC_001691), human papillomavirus type 53 (NC OO 1593), human papillomavirus type 60 (NC OO 1693), human papillomavirus type 63 (NC_001458), human papillomavirus type 6b (NC_OO1355), human papillomavirus type 7 (NCJ)Ol 595), human papillomavirus type 71 (NC_002644), human papillomavirus type 9 (NC_001596), human papillomavirus type 92 (NC 004500), human papillomavirus type 96 (NC_005134), human parainfluenza virus 1 (NC_003461), human parainfluenza virus 2 (NC 003443), human parainfluenza virus 3 (NC_001796), human parechovirus (NC_001897), human parvovirus 4 (NC_007018), human parvovirus Bl 9 (NC_000883), human respiratory syncytial virus (NC 001781) , human rhinovirus A (NC OOl 617), human rhinovirus B (NC OO 1490), human spumaretrovirus (NC_001795), human T-lymphotropic virus 1 (NC 001436), human T-lymphotropic virus 2 (NCJ)01488).

In certain examples, the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from an oncogenic virus, such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, e.g., HPV16, HPV 18). In other examples, the target protein produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is from a pathogenic virus, such as a Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or a Herpes Simplex Virus (HSV).

III. Sample Preparation

The tissue samples described herein can be prepared using any method known in the art. Generally, tissue samples are prepared by fixing and embedding the tissue in a medium.

In some examples an embedding medium is used. An embedding medium is an inert material in which tissues and/or cells are embedded to help preserve them for future analysis. Embedding also enables tissue samples to be sliced into thin sections. Embedding media include paraffin, celloidin, OCT™ compound, agar, plastics, or acrylics.

Many embedding media are hydrophobic, therefore the inert material may need to be removed prior to histological or cytological analysis, which utilizes primarily hydrophilic reagents. The term deparaffinization or dewaxing is broadly used herein to refer to the partial or complete removal of any type of embedding medium from a biological sample. For example, paraffin-embedded tissue sections are dewaxed using organic solvents, such as toluene, xylene, limonene, or other suitable solvents.

The process of fixing a sample can vary. Fixing a tissue sample preserves cells and tissue constituents in as close to a life-like state as possible and allows them to undergo preparative procedures without significant change. Fixation arrests the autolysis and bacterial decomposition processes which begin upon cell death, and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing, such as for IHC or in situ hybridization. Tissues can be fixed either by perfusion or by submersion in a fixative. Fixatives can be classified as cross-linking agents (such as aldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde cross-linking agents), oxidizing agents (e.g., metallic ions and complexes, such as osmium tetroxide and chromic acid), protein-denaturing agents (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid), microwaves, and miscellaneous fixatives (e.g., excluded volume fixation and vapor fixation). Additives may also be included in the fixative, such as buffers, detergents, tannic acid, phenol, metal salts (such as zinc chloride, zinc sulfate, and lithium salts), and lanthanum.

The most commonly used fixative in preparing samples for IHC is formaldehyde, generally in the form of a formalin solution (4% formaldehyde in a buffer solution, referred to as 10% buffered formalin). In one example, the fixative is 10% neutral buffered formalin. Sample preparation can also include using one or more cross-linking agents.

Cross linking agent are homo- or hetero-multifunctional reagents with at least two identical or non-identical groups that are reactive to functional groups present in

proteins, such as sulfhydryls and/or amine groups. In some examples, a protein cross-linker is amine reactive, meaning it is capable of forming a bond, typically a covalent bond, with an amine group, such as an amine group present in a protein, for example an amine group present on a lysine or arginine residue. Cross-linking agents which are used in histology to prepare tissue samples for IHC act by creating covalent bonds between cellular components in the sample, such as within or between proteins, within or between nucleic acids, or between proteins and nucleic acids. Cross-linking agents include aldehyde cross-linking agents and non-aldehyde cross-linking agents. Aldehyde cross-linking agents include formaldehyde, paraformaldehyde, glyoxal, glutaraldehyde, adipaldehyde, succinaldehyde, and suberaldehyde. In a particular example, the cross-linking agent comprises formaldehyde. Non-aldehyde cross-linking agents include cyanuric chloride, carbodiimides, diisocyanates, diimido esters, diethylpyrocarbonate, and maleimides.

IV. Probes

As described above, a probe includes a targeting moiety and a label. The targeting moiety functions to both specifically bind to a target molecule and associate with a label, such that the target is detectable. The targeting moiety can be associated with the label indirectly or directly. A person of ordinary skill in the art will appreciate that the label can be any of a variety of molecules that are known to a person of ordinary skill in the art, such as chromogenic molecules (e.g., molecules producing a pigment or coloring matter) or fluorophores (e.g., a molecule that absorbs a photon and triggers the emission of another photon with a different wavelength). In some examples, the chromogenic or fluorescent molecules are not detectable until they are reacted with an enzyme and/or an additional substrate. The label is used to detect or visualize the probe-target complex.

A targeting moiety can be associated with multiple labels. In some examples, a targeting moiety can be associated with at least about 2 labels, 3 labels, 4 labels, or at least about 5 labels. The increase in the number of labels associated with a targeting moiety will amplify the signal from a single target. In some examples, the labels can be different. For example, in some embodiments at least about one chromogenic label and at least about one fluorescent label can be

associated with a single targeting moiety. Used in this way, the target can be detected using either a brightfield microscope or a fluorescence microscope.

In certain embodiments multiple labels can be associated with a targeting moiety using haptens. Thus, detection can be facilitated by using anti-hapten monoclonal antibodies. FIG. 1 schematically represents one such embodiment. FIG. 1 shows a sample 10 that includes a particular target 14, such as a protein target, situated in a tissue 12. A primary antibody 18 directed to the target 14 is administered in a manner effective for the antibody to recognize the target. Primary antibody 18 has at least one hapten 50, and potentially plural haptens 50, conjugated thereto. A person of ordinary skill in the art will recognize first that the number of haptens conjugated to the antibody can vary. However, this number typically is from 1 to about 5 haptens, but more typically is 2 to 3. Furthermore, a person of ordinary skill in the art will appreciate that the haptens conjugated to the primary antibody can be the same or different. Sample 10 is treated with anti-hapten antibodies 52. For example, in the embodiment illustrated in FIG. 1 , haptens 50, conjugated to the primary antibody 18, then effectively become coupled to an anti-hapten antibody 52. One disclosed embodiment now involves treating the sample 10 with an antibody that recognizes the anti-hapten antibody. In the illustrated embodiment of FIG. 1, anti-antibodies 54 are conjugated to an enzyme that reacts with a label, such as the illustrated horseradish peroxidase (HRP) enzymes 56. This complex is then incubated with an HRP substrate, as is known to persons of ordinary skill in the art, to form detectable, e.g., colored, precipitates.

Targeting moieties can be designed to be directly conjugated to a label. Used in this way the targeting moiety/label complex (i.e., the probe) is contacted with the sample and the target is detected.

Targeting moieties can also be indirectly associated with a label. In some examples, a first targeting moiety is contacted with a sample. The targeting moiety can be either nucleic acid based or protein based. The targeting moiety can be conjugated to another moiety that is then bound for instance by a secondary antibody or a non-peptide based binding moiety, such as biotin. The secondary antibody or non-peptide binding pair can then be linked to a label. In another

example, a targeting moiety can be indirectly associated with a label by conjugating the targeting moiety, either directly or indirectly, to a peptide having enzymatic activity. The enzymatic activity is chosen so that upon addition of a substrate(s) the substrate(s) is converted into a label, or becomes a more active label. Examples of enzymatic activities that can be included in probes include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

Exemplary non-limiting examples of enzyme substrate pairs include: horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor (e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)); alkaline phosphatase (AP) withpαrα-nitrophenyl phosphate as chromogenic substrate; and beta-D-galactosidase (beta-D-Gal) with a chromogenic substrate (e.g., p- nitrophenyl-beta-D-galactosidase) or fluorogenic substrate (e.g., 4- methylumbelliferyl-beta-D-galactosidase). Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Patent Nos. 4,275,149 and 4,318,980. When a probe is made from the indirect association of one or more additional molecules, the additional molecules can be referred to probe components.

As mentioned above, in some examples the label is indirectly conjugated with an antibody. One of ordinary skill in the art will be aware of various techniques for achieving this. For example, the antibody can be conjugated to biotin and biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, as mentioned above to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten and a label is conjugated with an anti-hapten antibody. Thus, indirect conjugation of the label with the targeting moiety can be achieved.

When the probe includes an enzyme that reacts with a substrate to generate the label the label can be a chromagenic compound, fluorogenic compound, or luminogenic compound. There are numerous examples of such substrates. For example, many such compounds can be purchased from Invitrogen, Eugene Oregon. Particular non-limiting examples of chromogenic compounds include di- aminobenzidine (DAB), 4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3- ethylbenzothiazoline sulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo- 4-chloro-3-indolyl-β-galactopyranoside (X-GaI), methylumbelliferyl-β-D- galactopyranoside (MU-GaI), p-nitorphenyl-α-D-galactopyranoside (PNP), 5- bromo-4-chloro-3-indolyl- β -D-glucuronide (X-Gluc), 3-amino-9-ethyl carbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue and tetrazolium violet. Labeled secondary antibodies can be purchased from a number of commercial sources, such as, but not limited to, Pierce Co. Amersham and Evident Technologies provide a broad range of conjugated antibody possibilities. CyDye, EviTag Quantum Dot, fluorescein (FITC), and rhodamine can be utilized. These conjugates span a variety of applications, colors, and emission ranges. The EviTag Quantum Dots from Evident Technologies offer photo-stability and multicolor fluorescence in a variety of wavelengths, with the advantage over organic fluorophores of improved photostability, color multiplexing, and single source excitation. Each Evitag generates a sharp emission wavelength and therefore can be used for multiplexing in intact cell environments. The Amersham CyDyes offer photostability over a broad range of pH values.

For a tutorial on fluorescent markers, with the chemical structures of the labels, see: www.hmds.org.uk/fluorochrome.html. The following website provides additional information concerning on how to label with haptens: probes.invitrogen.com/handbook/boxes/2020.html. One type of molecule that can be used to indirectly link a label to a targeting moiety is a covalent conjugate of an antibody and a fiuorophore. Directing photons toward the conjugate that are of a wavelength absorbed by the fiuorophore

stimulates fluorescence (emission) that can be detected and used to qualitate, quantitate and/or locate the antibody.

Chromophoric and/or fluorescent semiconductor nanocrystals, also often referred to as quantum dots, can be used as labels. Nanocrystalline quantum dots are semiconductor nanocrystalline particles, and without limiting the disclosure to use with particle light emitters of a particular size, typically measure 2-10 nm in size (roughly the size of typical proteins). Quantum dots typically are stable fluorophores, often are resistant to photo bleaching, and have a wide range of excitation, wave-length and narrow emission spectrum. Quantum dots having particular emission characteristics, such as emissions at particular wave-lengths, can be selected such that plural different quantum dots having plural different emission characteristics can be used to identify plural different targets. Quantum dot bioconjugates are characterized by quantum yields comparable to the brightest traditional dyes available. Additionally, these quantum dot-based fluorophores absorb 10-1000 times more light than traditional dyes. Emission from the quantum dots is narrow and symmetric, which means overlap with other colors is minimized, resulting in minimal bleed through into adjacent detection channels and attenuated crosstalk, in spite of the fact that many more colors can be used simultaneously. Symmetrical and tuneable emission spectra can be varied according to the size and material composition of the particles, which allows flexible and close spacing of different quantum dots without substantial spectral overlap. In addition, their absorption spectra are broad, which makes it possible to excite all quantum dot color variants simultaneously using a single excitation wavelength, thereby minimizing sample autofluorescence. Furthermore, pegylation, the introduction of polyethylene glycol groups onto the quantum dot conduits, can substantially decrease non-specific protein:quantum dot interaction. Certain quantum dots are commercially available, such as from Quantum Dot Corp., of Hayward, California, and Invitrogen.

Additional examples of labels that can be used in the methods provided herein include those provided in Table 1.

Table 1: Labels

In some embodiments a sample can be labeled sequentially with a fluorescent label then a chromogenic label, or the sample can be labeled simultaneously with both a fluorescent and a chromogenic label. As used herein, "simultaneous" means that at one point in time the sample includes both a fluorescent label and a chromogenic label.

In some examples a targeting moiety that is conjugated to a peptide having peroxidase activity is contacted with the sample. A first label that interacts with the peroxidase is added to the sample and the label is deposited at the site of the target. A second label can then be added that interacts with peroxidase as well and the second label is then also deposited at the site of the target. Thus, the sample is simultaneously labeled with both labels and these labels can have different physical properties. For example, one label can be chromogenic and one label can be fluorescent. One of ordinary skill in the art will appreciate that any fluorescent and chromogenic label that interacts with the same enzymatic activity can be used. Exemplary, non-limiting examples of substrates that can be used as a first label in this method include tyramide derivatives, such as fluorescyl tyramide, tetramethyl- rhodamine tyramide, cyanine-3 -tyramide, cyanine-5 -tyramide and combinations thereof. Exemplary second labels include, for example, chromogenic molecules that interact with peroxidases such as 4-chloro-l-naphthol ("4CN"), 3-amino-9-ethyl- carbazole ("AEC"), and 3,3'- diaminobenzidine ("DAB") and its derivatives.

In some examples the tissue sample can be labeled with a first fluorescent label and then that label can be converted to a chromogenic label. Digital images can be taken of the sample contacted with the first label and overlaid with a digital image of the sample contacted with the second label.

V. Contacting Probes with Samples

The sample can be prepared using any method known in the art. The samples are then placed in contact with one or more probes. As will be appreciated by one of ordinary skill in the art, depending upon the nature of the probe (e.g., indirect or directly linked to one or more labels) the contacting step may actually include several different cycles of contacting the sample with mixtures that include

one or more probe components. For example, when the targeting moiety is not directly linked to a label, the targeting moiety can be contacted with a sample, then the secondary antibody or non-peptide based binding moiety can be contacted with the sample. In some examples, the sample can then be contacted with substrates that interact with enzymatic activities associated with the targeting moiety. As one of ordinary skill in the art will appreciate the various components that eventually form the probe can be added individually or simultaneously to the sample.

In a more specific, but yet general description the samples can be placed on slides that are deparaffinized by placing the slides in an appropriate buffer, such as a citrate buffer. The buffer and slide can optionally be heated. The slides including the samples can then be rinsed in distilled water. Following the rinse, slides can be blocked and washed.

As described above, the sample is then contacted with the probe, or through subsequent steps placed in contact with the various components of the probe such that the probe is eventually formed and is associated with the target. When using an antibody-based binding moiety several different concentrations of binding moiety can be tried to identify the concentration needed for detection of the target. For example, a dilution of from about 1 : 100 to about 1 :25 of the antibody can be used. Similarly, when using a nucleic acid-based targeting moiety different concentrations and nucleic acid sequences can be tested to determine the targeting moiety that gives the desired result. Control slides can also be prepared that are not contacted with the targeting moiety. Upon binding of the probe to the target the sample can be detecting using the appropriate imaging device.

VI. Imaging

Certain aspects, or all, of the disclosed embodiments can be automated, and facilitated by computer analysis and/or image analysis system. In some applications precise color ratios are measured. Certain disclosed embodiments involve acquiring digital images. This can be done by coupling a digital camera to a microscope. Digital images obtained of stained samples are analyzed using image analysis software. Color can be measured in several different ways. For example, color can be measured as red, blue, and green values; hue, saturation, and intensity values; and

by measuring a specific wavelength or range of wavelengths using a spectral imaging camera.

One disclosed embodiment involves using brightfield imaging with chromogenic dyes. White light in the visible spectrum is transmitted through the dye. The dye absorbs light of certain wavelengths and transmits other wavelengths. This changes the light from white to colored depending on the specific wavelengths of light transmitted. The chromogenic dye also may be a fluorogenic dye visualized using a fluorescence microscope.

Standard fluorescence microscopes can be used to detect quantum dot bioconjugates. Since quantum dot conjugates are virtually photo-stable, time can be taken with the microscope to find regions of interest and adequately focus on the samples.

As an example, fluorescence can be measured with the multispectral imaging system Nuance™ (Cambridge Research & Instrumentation, Woburn, MA). As another example, fluorescence can be measured with the spectral imaging system SpectrView™ (Applied Spectral Imaging, Vista, CA). Multispectral imaging is a technique in which spectroscopic information at each pixel of an image is gathered and the resulting data analyzed with spectral image-processing software. For example, the Nuance system can take a series of images at different wavelengths that are electronically and continuously selectable and then utilized with an analysis program designed for handling such data. The Nuance system is able to obtain quantitative information from multiple dyes simultaneously, even when the spectra of the dyes are highly overlapping or when they are co-localized, or occurring at the same point in the sample, provided that the spectral curves are different. Many biological materials autofluoresce, or emit lower-energy light when excited by higher-energy light. This signal can result in lower contrast images and data. High- sensitivity cameras without multispectral imaging capability only increase the autofluorescence signal along with the fluorescence signal. Multispectral imaging can unmix, or separate out, autofluorescence from tissue and, thereby, increase the achievable signal-to-noise ratio.

When digital imaging is used in combination with multiplexing, an image can be captured of a first set of probes that have been used to target multiple targets

in a sample. This process can be described as capturing a first data set. The probes can be released from the sample as described herein and a second set of probes can be used to identify targets in the sample. Some or all of the labels used on the first set of probes and the second set of probes can be the same, or they can be different. When the labels are the same the digital image of one or both of the sets can be altered such that color assignments (e.g., a blue pixel in the first data set can be assigned a different color and the same label reused in the second set of probes) are changed for the matching labels, thus allowing for the digital images to be overlaid without the loss of information. The samples can also be evaluated qualitatively and semi-quantitatively.

Qualitative assessment includes assessing the staining intensity, identifying the positively-staining cells and the intracellular compartments involved in staining, and evaluating the overall sample or slide quality. Separate evaluations are performed on the test samples and this analysis can include a comparison to known average values to determine if the samples represent an abnormal state.

VII. Multiplexing Probes

Multiplexing probes involves using more than one probe to detect more than one target in a tissue sample. The labels used in the probes can have physical characteristics that are distinguishable from each other and that do not quench the signal from each other.

In some examples, probes are used in a multiplex scheme that includes targeting moieties that contain protein (e.g., peptide-based probes). After the first set of more than one probe is analyzed the first set can be released from the tissue sample. The probes can be heated to a temperature sufficient to release the probe from the target, but yet not damage the tissue sample so that it can be re-probed. One of ordinary skill in the art will appreciate that the temperature used will depend upon the individual probe and target combination, but generally temperatures from about 50° C to about 99° C will allow the probe to be substantially released from the target while not causing substantial damage to the sample. In some examples, the sample is heated to a temperature of less than 1 10° C, such as less than 100° C, for example from about 50° C to about 90° C, from about 65° C to about 85° C or from

about 70° C to about 80° C, for example for a period of time of at least 3 minutes, at least 5 minutes or at least 10 minutes, such as from about 3 to about 30 minutes.

One of ordinary skill in the art can determine the temperature and duration of exposure to heat that is necessary for a specific probe/target combination. For example, a first probe set can be used to detect a target in a sample and then the sample can be exposed to 15 minutes of 65° C heat and the sample can be viewed under a brightfield microscope if the first probe set was chromogenic, or under a fluorescent microscope if the first probe set was fluorescent. If, after the heating step, the signal from the first probe set is still strong then the sample can be washed for a longer period of time and/or at a higher temperature.

In one example, the sample is contacted with a solution during the heating process. The solution can contain agents, such as water, buffers, detergents, and/or chelating agents. In some examples, the solution includes water, such as at least about 2% water, or at least about 5%, 10%, 20%, 30%, 40%, 50%, or at least about 75% water. In further examples, the solution includes at least one buffering agent, such as Tris, Tris buffered saline, phosphate buffered saline, citrate, borate, acetate, or combinations thereof.

VIII. Counterstaining Counterstaining is a method of post-treating the samples after they have already been stained with agents to detect one or more targets, such that their structures can be more readily visualized under a microscope. For example, a counterstain is optionally used prior to coverslipping to render the immunohistochemical stain more distinct. Counterstains differ in color from a primary stain. Numerous counterstains are well known, such as hematoxylin, eosin, methyl green, methylene blue, Geimsa, Alcian blue, and Nuclear Fast Red.

In certain examples methods of counterstaining include counterstaining using complementary counterstains. Complementary counterstains are stains that can be used with fluorescent probes to facilitate the detection of cellular structures at the same time that the probe is being detected. Therefore, complementary counterstains are chosen based upon the similarity of their absorbance wavelength and difference of their emission wavelength qualities to the probe's absorbance and emission

qualities. Generally, complementary counterstains display a peak absorbance that is within 100 nm of the peak absorbance of the probe. In other examples, the counterstain displays a peak absorbance that is within 90, 80, 70, 60, 50, 40, 30, 20, or 10 nm of the peak absorbance of the probe. Similarly, in some examples, complementary counterstains will display a peak emission wavelength that is at least about 100 nm different from the peak emission wavelength of the probe. In other examples, the counterstain displays a peak emission wavelength that is at least about 90, 80, 70, 60, 50, 40, 30, 20, or 10 nm different from the peak emission wavelength of the probe. In some examples, a tissue sample is stained with both a chromogenic label and a fluorescent label and when a complementary counterstain is used it can provide cell structure information in both brightfield microscopy and fluorescent microscopy.

Exemplary, non-limiting examples of complementary counterstains are provided in Table 2, below.

Table 2 Exemplary Complementary Counterstains

In some examples, more than one stain can be mixed together to produce the counterstain. Thus, providing flexibility and the ability to choose a first stain for the mixture that has a particular attribute, but yet does not have a different desired attribute and then add a second stain that displays the missing desired attribute to the mixture. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed together to form a counterstain.

EXAMPLES

Example 1 This example describes one embodiment of a method for preparing slides containing a sample for immunohistochemistry analysis, for example to probe and re-probe the sample. One of ordinary skill in the art will appreciate that variations on this method as well as alternative methods can be used.

Samples are deparaffinized and rehydrated as follows. The sample is washed three times for 5 minutes in xylene; two times for 5 minutes in 100% ethanol; two times for 5 minutes in 95% ethanol; and once for 5 minutes in 80% ethanol.

The samples are blocked by placing them in endogenous blocking solution (methanol+2% hydrogen peroxide) for 20 minutes at room temperature followed by rinsing twice for 5 minutes each in deionized water and then twice for 5 minutes in phosphate buffered saline (PBS), pH 7.4.

Optionally the sample can be subjected to antigen retrieval protocols. The sample can also be blocked and stained by incubating them with PBS/1% bovine serum albumin (PBA) for 1 hour at room temperature. The samples are covered with primary antibody diluted in PBA, and placed in the lid of the humidity chamber and incubated either at room temperature for 1 hour or overnight at 4° C. The samples are subsequently rinsed in PBS and covered with secondary antibody in PBA for 30 minutes- 1 hour at room temperature in the humidity chamber. The samples are then rinsed again in PBS. The samples are then incubated PBS for 1 hour at room temperature in the humidity chamber. The samples are then rinsed again in PBS. A digital image can be obtained of the sample.

The samples are then heated to a temperature of from about 50° C-90° C for 3-15 minutes, rinsed in PBS, and a second set of peptide-based probes is used to identify a second set of targets. A digital image can be obtained of the sample. A counterstain can be added, for example using 0.01% Light Green acidified with 0.01% acetic acid for 1-2 minutes depending on intensity of counterstain desired.

Example 2

This examples concerns tonsil tissue that was probed and then heated to release the first probe and then reprobed. Tonsil tissues were fixed in 10% neutral buffered formalin for 12 hours.

The fixed tonsils were then dehydrated in graded alcohols, cleared in xylene, and embedded in paraffin blocks using standard histologic methods. Sections were cut from the paraffin blocks at a thickness of four microns and attached to microscope slides. The slides with attached tissue sections were deparaffinized in xylene, and rehydrated through alcohols to water. The slides were then rinsed with phosphate buffered saline, and then exposed to antigen retrieval by heating the slides in 0.1 M citrate buffer, pH 6.0, for 10 minutes at 97° C.

Slides were stained with rhodamine conjugated anti-kappa for 30 minutes at room temperature. Slides were rinsed in water, and temporarily coverslipped using 50% glycerol in water as the mounting medium. Photomicrographs were captured at 2OX magnification (FIG. 2A).

Slides were then simultaneously de-coverslipped and stripped of antibody by heating the slides to 60° C for five minutes. The slides were then rinsed in phosphate buffered saline and stained at room temperature for 30 minutes with fluorescein conjugated anti-lamba antibody. Slides were rinsed in water, and temporarily coverslipped using 50% glycerol in water as the mounting medium. Photomicrographs were captured at 2OX magnification (FIG. 2B).

Slides were then simultaneously de-coverslipped and stripped of antibody by heating the slides to 60° C for five minutes. The slides were then rinsed in phosphate- buffered saline and stained at room temperature with Alexa 647 conjugated anti-cytokeratin for 30 minutes at room temperature. Slides were rinsed in water, and mounted with 50% glycerol in water as the mounting medium. Photomicrographs were captured at 2OX magnification (FIG. 2C).

The resulting images can be combined as shown in FIGS. 3A-C. These results show that antibodies bound to tissues can be stripped using a mild heat treatment of 60° C for 5-10 minutes. Following stripping tissues can be stained using a second antibody to another molecular target. The process of

stripping and restaining can be performed multiple times. Digital images of the individual antibodies can be manipulated to by computer to separate or combine images in any combination. Using image analysis methods permits quantification of the amount of signal associated with molecular target and to compare ratios of the different molecular targets. Furthermore, the color of the fluorochrome can be digitally assigned to a new color. In this way the same fluorochrome could be used in different cycles and its color digitally changed so that its new color can be differentiated from the same fluorochrome used in a previous cycle. Therefore, the number of molecular targets that can be stained is not limited by the number of fluorochromes available.

Having illustrated and described the principles of the disclosure in multiple embodiments and examples, it should be apparent that the disclosure can be modified in arrangement and detail without departing from such principles. The disclosure encompasses all modifications coming within the spirit and scope of the following claims: