Use of single-cell ELISA starting from deparaffinized cells to detect molecules of interest

Abstract

Provided herein is a method for detecting the presence of a molecule in a sample, such as a patient's bodily fluid or tissue, comprising: a. obtaining cells from the sample; b. treating the cells with a fixative; c. paraffin-embedding the fixed cells; d. deparaffinizing and suspending the cells to obtain a single cell suspension; e. contacting the suspended cells with a first detection agent that binds to at least one molecule in the suspended cells, and contacting the cells bound to the first detection agent with a second detection agent; and f. detecting the presence of the second detection agent bound to the cells of the sample, wherein detection of an above background amount of the second detection agent bound to the sample indicates the presence of the at least one molecule in the sample.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Patent Application Publication No. 63 / 214,177, filed June 23, 2021, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION Provided herein are methods for detecting the presence of a molecule in a sample, such as a bodily fluid or tissue, from a patient. Summary of the Invention [Means for solving the problem]

[0003] In one aspect, provided herein is a method of detecting a molecule in a sample, comprising obtaining cells from the sample, treating the cells with a fixative, paraffin-embedding the fixed cells, deparaffinizing and suspending the cells to obtain a single cell suspension, contacting the suspended cells with a first detection agent that binds to at least one molecule of the suspended cells, contacting the cells bound to the first detection agent with a second detection agent, and detecting the presence of the second detection agent bound to the cells of the sample, wherein detection of an above background amount of the second detection agent bound to the sample indicates the presence of the at least one molecule in the sample.

[0004] In some embodiments, the molecule is a nucleic acid or a protein. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is DNA.

[0005] In some embodiments, the method further comprises removing unbound cells after contacting the suspended cells with a first detection agent, hi some embodiments, the method further comprises removing unbound second detection agent after contacting the cells bound to the first detection agent with a second detection agent.

[0006] In some embodiments, the fixative is selected from the group including formaldehyde, paraformaldehyde, glutaraldehyde, or neutral buffered formalin. In some embodiments, the fixative is neutral buffered formalin. In some embodiments, the neutral buffered formalin is 10% neutral buffered formalin.

[0007] In some embodiments, the step of treating the cells with the fixative lasts for about 30 minutes to 60 minutes, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 7 hours, 7 hours to 8 hours, 8 hours to 9 hours, 9 hours to 10 hours, 10 hours to 11 hours, 11 hours to 12 hours, 12 hours to 13 hours, 13 hours to 14 hours, 14 hours to 15 hours, 15 hours to 16 hours, 16 hours to 17 hours, 17 hours to 18 hours, 18 hours to 19 hours, 19 hours to 20 hours, 20 hours to 21 hours, 21 hours to 22 hours, 22 hours to 23 hours, 23 hours to 24 hours, 24 hours to 36 hours, or 36 hours to 48 hours. In some embodiments, the step of treating the cells with the fixative is performed at 4°C, room temperature, 40°C, or 60°C. In some embodiments, treating the cells with a fixative is carried out at room temperature for 24 hours.

[0008] In one aspect of the methods provided herein, paraffin embedding of the fixed cells comprises contacting the cells with ethanol, contacting the cells with xylene, and incubating the cells with paraffin.

[0009] In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 70% ethanol in water for 30 minutes, contacting the cells with 80% ethanol in water for 30 minutes, contacting the cells with 95% ethanol in water for 30 minutes, and contacting the cells with 100% ethanol for 30 minutes.

[0010] In some embodiments, contacting the cells with xylene comprises three changes of xylene for 20 minutes each.

[0011] In some embodiments, incubating the cells with paraffin comprises four changes of paraffin for 20 minutes each. In some embodiments, incubating the cells with paraffin is performed at 60°C.

[0012] In some embodiments, the cells are deparaffinized by contacting the cells with xylene, hi some embodiments, the cells are contacted with xylene for about 5-10 minutes, 10-15 minutes, 15-20 minutes, 20-30 minutes, 30-60 minutes, 60-90 minutes, or 90-120 minutes.

[0013] In some embodiments, the cells are further contacted with a continuous ethanol gradient, including contacting the cells with 70% ethanol in water for about 15 to about 30 minutes, contacting the cells with 95% ethanol in water for about 15 to about 30 minutes, and finally contacting the cells with 100% ethanol for about 15 to about 30 minutes.

[0014] In one aspect of the methods provided herein, the cells are resuspended in an antigen unmasking solution.

[0015] In some embodiments, the cells are further heated for about 30 minutes at 95° C. In some embodiments, the cells are heated by microwave radiation.

[0016] In some embodiments, the first and / or second detection agent is an antibody or antigen-binding fragment thereof. In some embodiments, the first and / or second detection agent is an RNA-based binder molecule.

[0017] In one aspect of the methods provided herein, the sample comprises cells from a bodily fluid or tissue. In some embodiments, the bodily fluid is blood, serum, or plasma. In some embodiments, the sample is from a patient. In some embodiments, the patient is a mammal. In some embodiments, the mammal is a human. In some embodiments, the sample comprises cells from an immortalized cell line.

[0018] In another aspect, provided herein is a kit for carrying out the method of any one of the embodiments provided herein.

[0019] In another aspect, provided herein is a sample of cells prepared by the method of any one of the embodiments provided herein. [Brief description of the drawings]

[0020] The foregoing Summary of the Invention, as well as the following Detailed Description of certain embodiments of the present application, will be better understood when read in conjunction with the accompanying drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Figure 1] FIG. 1 shows a schematic diagram of the methods provided herein for enhanced molecular detection and screening. [Diagram 2] Antibody binding in the form of optical density measured at 450 nm is shown. Each antibody binding was tested in duplicate with 30,000 cells per well. The condition named "control negative" involved cells incubated with antibody diluent only. [Figure 3A] The results of immunohistochemistry ("IHC") assays performed with various antibody solutions on 4 um sections of cell pellet blocks are shown. Figure 3A shows the IHC cell pellet staining for each antibody. Figure 3B shows the H-score for each stained pellet. The H-score for each stained pellet was calculated based on an area quantification algorithm that calculates the total surface intensity staining of the cell pellet. [Figure 3B] The results of immunohistochemistry ("IHC") assays performed with various antibody solutions on 4 um sections of cell pellet blocks are shown. Figure 3A shows the IHC cell pellet staining for each antibody. Figure 3B shows the H-score for each stained pellet. The H-score for each stained pellet was calculated based on an area quantification algorithm that calculates the total surface intensity staining of the cell pellet. [Figure 4] Correlation assessment between the ELISA-like method provided herein and IHC staining is shown. The Pearson correlation coefficient r=0.929 (GraphPad Prism) indicated sufficient comparability between the screening results obtained by the high-throughput ELISA-like method and conventional IHC performed on formalin fixed paraffin embedded ("FFPE") tissue samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Various publications, articles and patents are cited or described in the "Background" and throughout the specification, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms used herein have the meanings set forth herein.

[0023] The techniques and procedures described or referenced herein include those generally well understood by those skilled in the art and / or those commonly employed by those skilled in the art using conventional techniques, such as the widely used techniques described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001), Current Protocols in Molecular Biology (Ausubel et al. eds., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2d ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For the purposes of interpreting this specification, the following explanations of terms apply, and where appropriate, terms used in the singular form also include the plural form and vice versa. In the event that any explanation of a term provided herein conflicts with any document incorporated by reference, the explanation of the term provided below shall prevail.

[0024] The following references are incorporated by reference in their entireties: U.S. Patent Application Publication No. 10 / 872,462, U.S. Patent Application Publication No. 14 / 115,327, U.S. Patent Application Publication No. 13 / 536,021, U.S. Patent Application Publication No. 10 / 320,219, U.S. Patent Application Publication No. 11 / 319,118, U.S. Patent Application Publication No. 14 / 652,407, U.S. Patent Application Publication No. 13 / 571,854, U.S. Patent Application Publication No. 11 / 772,288, McGinnis et al. (Journal of Pathology; 2021; 254(4); 405-417), Sun et al. (PLoS ONE; 16(2)e0247238; 2021), Gentles et al. (Journal of Clinical Pathology 2021;74:469-474), Wilgenbusch et al., 2020 (Journal of the American Society of Cytopathology; 9,20-25), and Mairaville et al. (Antibodies; 2021, 10, 4).

[0025] 5.1.Definition It should be noted that as used herein in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0026] In the event that there are a plurality of definitions for terms herein, those in this section prevail unless stated otherwise.

[0027] The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" refers to within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" refers to within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0028] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0029] It will be understood that as used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to be inclusive of the stated element or elements, but not to the exclusion of other elements or elements, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or that are inherent to such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0030] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be within the meaning and thus meets the requirements of the term "and / or."

[0031] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout the specification and claims, includes any listed element or elements, but indicates that no additional element or elements are added to the specified method, structure, or composition.

[0032] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, indicates the inclusion of any recited element or group of elements, optionally including any recited element or group of elements that do not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP § 2111.03.

[0033] As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance to a patient, such as by oral, mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or a symptom thereof. When a disease or a symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease or a symptom thereof.

[0034] As used herein, the term "polynucleotide" is also referred to interchangeably as "nucleic acid molecule," "nucleotide," or "nucleic acid," and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions that include RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contain one or more modified bases, and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. A variety of modifications can be made to DNA and RNA. Thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms having characteristics of viral and cellular DNA and RNA. "Polynucleotide" also includes relatively short nucleic acid strands, often referred to as oligonucleotides.

[0035] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes transcription of a gene into RNA. The term also includes translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications. The expressed antibody may be present in the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or may be anchored to the cell membrane.

[0036] As used herein, the term "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0037] The peptide sequences described herein are written according to the usual convention with the N-terminal region of the peptide on the left and the C-terminal region on the right, although isomeric forms of amino acids are known, unless expressly indicated otherwise, it is the L-form of the amino acid that is shown.

[0038] The terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and are used in the broadest sense, specifically including, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single chain antibodies, single domain antibodies (e.g., VHH), and fragments thereof (e.g., domain antibodies). Antibodies may be human, humanized, chimeric, and / or affinity matured, and may also be antibodies from other species, e.g., mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides capable of binding to a specific molecular antigen and composed of two identical paired polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the respective amino-terminal portions of each chain containing a variable region of about 100 to about 130 or more amino acids, and the respective carboxy-terminal portions of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies such as those derived from Camelidae species (e.g., llamas and alpacas) or humanized variants thereof, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above (e.g., antigen-binding fragments), and refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which the fragment is derived.Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single chain Fvs (scFv) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules that contain an antigen-binding site that binds an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibodies can be agonist or antagonist antibodies. The antibody may be neither an agonist nor an antagonist.

[0039] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0040] "Antigen-binding domain" or "antigen-binding fragment" or "domain that binds to antigen" refers to a portion of a molecule that specifically binds to an antigen. An antigen-binding domain may include a portion of an immunoglobulin that binds to an antigen, such as a heavy chain variable domain (VH), a light chain variable domain (VL), VH and VL, Fab, Fab', F(ab')2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH or one VL, shark variable IgNAR domain, camelized VH domain, VHH, a minimal recognition unit consisting of amino acid residues that mimic the CDRs of an antibody such as a FR3-CDR3-FR4 portion, HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, and a non-antibody scaffold that binds to an antigen.

[0041] As used herein, "epitope" is a term of the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single-chain antibody sequence) can specifically bind. An epitope can be a linear or conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of a polypeptide (a "linear" epitope), or an epitope can include amino acids from two or more non-contiguous regions of a polypeptide (a "conformational", "non-linear", or "discontinuous" epitope). In general, it will be understood by those skilled in the art that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids, regardless of whether the amino acids are folded into a native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues that make up the epitope exhibit a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0042] An "intact" antibody is one that contains an antigen-binding site as well as the constant domain of the light chain (CL) and at least the heavy chain constant regions CH1, CH2, and CH3. The constant region may include a human constant region or an amino acid sequence variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0043] A "single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a description of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0044] As used herein, a "single domain antibody" or "sdAb" refers to a single monomeric variable antibody domain capable of antigen binding. Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally lacking light chains, such as those from Camelidae species (e.g., llamas), single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. For example, single domain antibodies may be derived from antibodies produced in Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco, as described herein. Other species outside of Camelidae may also produce heavy chain antibodies that do not naturally have light chains. VHHs derived from such other species are within the scope of this disclosure. In some embodiments, the single domain antibodies (e.g., VHH) provided herein have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., a drug) as described herein. Single domain antibodies may be part of a larger binding molecule (e.g., a multispecific antibody or a functional exogenous receptor).

[0045] The term "binding" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site of an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The ratio (koff / kon) of the dissociation rate (koff) and the association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both kon and koff. The dissociation constant KD of the antibodies provided herein can be determined using any method provided herein or any other method known to one of skill in the art. Affinity at one binding site does not necessarily reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, comes into contact with an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reaction at a second site. The strength of multiple interactions between such a multivalent antibody and the antigen is called avidity.

[0046] As used herein, the term "body fluid" or "bodily fluid" refers to a fluid obtained from a patient, such as a mammalian (e.g., human) patient. For example, the body fluid can be blood, cerebral spinal fluid (CSF), breast milk, or urine. The body fluid can also be blood that has been fractionated to remove cells (i.e., plasma) or blood that has been fractionated to remove cells and clotting factors (i.e., serum).

[0047] As used herein, the term "capture moiety" or "first antibody" refers to a composition that can be specifically bound by another composition immobilized, e.g., bound, or otherwise linked to a solid support. Many of the detection moieties provided herein can also be used as capture moieties, so long as a binding event is involved. For example, useful capture moieties include affinity labels for which specific and selective ligands are available (e.g., biotin and avidin, glutathione and GST), haptens and proteins for which antisera or monoclonal antibodies are available (e.g., c-Myc), nucleic acid molecules with sequences complementary to the target, and peptides for which specific and selective ligands are available (e.g., histidine tags and Ni). Molecules that affect the binding properties to a chromatographic resin are also contemplated. The solid support can be, for example, a filter, a plate, a membrane, a chromatographic resin, or a bead.

[0048] As used herein, the term "cut-point factor" or "threshold" generally refers to a value used to mathematically manipulate the signal from a naive pool matrix (e.g., serum or plasma) to set the minimum signal from a sample required to be considered positive.

[0049] The term "derivative," as used in reference to antibody agents and polypeptides used in the methods provided herein, refers to polypeptides that have been chemically modified by techniques including, but not limited to, ubiquitination, conjugation to therapeutic or diagnostic agents, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (i.e., derivatization with polyethylene glycol), and chemically synthesized insertion or substitution of amino acids such as ornithine that do not normally occur in human proteins. Derivatives can retain the binding properties of the non-derivatized molecule.

[0050] As used herein, the terms "detectable moiety," "detection moiety," or "label" refer to a composition (e.g., a polypeptide or antibody) that is detectable by means including, but not limited to, spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful detectable moieties or labels include ruthenium (Ru)-based catalysts, europium, 32 P, 35 Detectable moieties or labels include, for example, fluorescent dyes, electron-dense reagents, enzymes (such as those commonly used in ELISA), biotin-streptavidin, dioxygenin, haptens and proteins for which antisera or monoclonal antibodies are available, and nucleic acid molecules having a sequence complementary to a target. Detectable moieties or labels often generate a measurable signal, such as a radioactive, colorimetric, luminescent, or fluorescent signal, that can be used to quantify the amount of bound detectable moiety or label in a sample.

[0051] As used herein, the term "detectable antibody" refers to any antibody that can be detected. In some embodiments, the antibody is directly labeled with a detectable moiety. In certain embodiments, the antibody is a detectable anti-Ig antibody. As used herein, the term "detectable anti-Ig antibody" refers to an anti-Ig antibody that can be detected. In some embodiments, the anti-Ig antibody is directly labeled with a detectable moiety in addition to its inherent binding to an Ig molecule. The Ig antibody can be, for example, of the IgG, IgE, IgM, IgD, IgA, or IgY isotype.

[0052] As used herein, the term "primary antibody" refers to an antibody that directly binds to an antigen of interest. As used herein, the term "secondary antibody" refers to an antibody that is conjugated to a detection label. In some embodiments, the secondary antibodies provided herein directly bind to the primary antibody. In other embodiments, the secondary antibodies provided herein indirectly bind to the primary antibody, for example, by binding to another antibody that recognizes the primary antibody.

[0053] As used herein in the context of a peptide or polypeptide, the term "fragment" refers to a peptide or polypeptide that comprises less than the full-length amino acid sequence. Such fragments can result, for example, from truncations at the amino terminus, truncations at the carboxy terminus, and / or internal deletion of a residue(s) from the amino acid sequence. Fragments can result, for example, from alternative RNA splicing or in vivo protease activity. Any fragment of a peptide or polypeptide disclosed herein is functional. In certain embodiments, a fragment comprises a polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of an amino acid sequence of an antibody that immunospecifically binds to a target antigen. In certain embodiments, an antibody fragment that immunospecifically binds to a target antigen retains at least one, at least two, or at least three functions of an antibody.

[0054] The term "identical" or percent "identity" in the context of two or more polynucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence, as determined using a sequence comparison algorithm or by visual inspection.

[0055] The term "antibody that immunospecifically binds" to a target antigen and similar terms are used interchangeably herein and refer to antibodies and fragments thereof that specifically bind only to a target antigen or epitope. In yet other embodiments, the antibodies provided herein immunospecifically bind to Ig, such as IgG, IgE, IgM, IgD, IgA isotypes.

[0056] As used herein, the term "interference" generally refers to the presence of a substance in a bodily fluid (e.g., serum or plasma) sample that prevents accurate detection and measurement of a target analyte. As used herein, interference generally refers to the effect of free drug or the effect of the matrix (e.g., serum or plasma) on the concentration-response relationship. For example, interference from the matrix may be evaluated relative to a sample without potential interference to target a range of relative accuracy of 75-125%.

[0057] The term "in vivo" in the context of a sample refers to a biological sample, e.g., a sample obtained from a subject, e.g., a patient, e.g., a human patient, including a biological or bodily fluid, e.g., blood, plasma, serum, bone marrow, cerebrospinal fluid, cerebral fluid, or tissue, e.g., lymphatic tissue, thin layer cytological samples, fresh frozen tissue samples, or tumor tissue. The term "in vivo" should be distinguished from the term "in vitro," which includes cells or cell lines cultured or grown outside a living organism, or biomolecular components of cells.

[0058] The terms "limit of detection," "LOD," or "sensitivity," as used herein, generally refer to the lowest analyte concentration in a bodily fluid (e.g., serum or plasma) sample that can be detected but not necessarily quantified as an exact value. For example, the LOD can be defined as the analyte concentration that consistently produces a signal greater than the measured mean response of the pooled naive matrix plus the cutpoint coefficient.

[0059] As used herein, the term "matrix" or "matrices" generally refers to the biological background in which an antibody is measured. Examples of matrices include, for example, body fluids and tissues.

[0060] The term "monoclonal antibody" refers to an antibody obtained from a homogeneous or substantially homogeneous population of antibodies, with each monoclonal antibody typically recognizing a single epitope on an antigen. In certain embodiments, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell, and the antibody immunospecifically binds only to an enzyme, as determined, for example, by ELISA or other antigen-binding or competitive binding assays known in the art. The term "monoclonal" is not intended to be limited to a particular method for making the antibody. For example, the monoclonal antibodies used in the methods provided herein can be made by the hybridoma method described in Kohler et al.; Nature, 256:495 (1975), or isolated from a phage library using techniques known in the art. Other methods for preparation of clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York).

[0061] As used herein, "polyclonal antibody" refers to an antibody population generated in an immunogenic response to a protein with many epitopes, and thus includes a variety of different antibodies directed to the same and different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York).

[0062] As used herein, the term "precision" generally refers to the variability of the signal between analysts and days. For example, precision can be assessed as the coefficient of variation, range of values, or using ANOVA statistics.

[0063] As used herein, the terms "prevent," "preventing," and "prevention" refer to the total or partial inhibition of the occurrence, recurrence, development, or spread of a disease and / or its associated symptoms (e.g., a disease or its associated symptoms associated with elevated phenylalanine levels, such as PKU or cancer in a patient) resulting from the administration of a therapy or combination of therapies provided herein.

[0064] As used herein, the term "reagent stability" generally refers to the robustness of a reagent preparation and storage stability. For example, reagent stability may be established by conditions that still allow values ​​to be measured within 75-125% accuracy for a freshly prepared reagent.

[0065] As used herein, the term "robustness" generally refers to the ability of an assay to remain unaffected by small variations in method parameters and indicates the reliability of the assay during normal running conditions. For example, robustness can be assessed as the percent change in reagent concentration, reagent volume, or incubation time that still produces a signal within 75-125% accuracy relative to nominal conditions.

[0066] As used herein, the term "sample" generally refers to a test fluid or tissue, e.g., taken from a patient, that can be used in the methods provided herein. In some embodiments, the sample is an in vivo sample, e.g., a bodily fluid (or biological fluid) from a subject, e.g., a patient, e.g., a human patient. Non-limiting examples of such bodily fluids include blood (e.g., human peripheral blood (HPB)), blood lysate, serum, plasma, fine needle aspirate, ductal lavage, cerebrospinal fluid, brain fluid, bone marrow, ascites, or any combination thereof. In other embodiments, the sample is taken from a biopsy tissue, such as a tumor tissue from a subject, or a thin layer cytological sample of other body tissues or organs. In certain embodiments, the sample comprises a peripheral blood sample, a tumor tissue or suspected tumor tissue, a thin film cytology sample, a fine needle aspirate sample, a bone marrow sample, a lymph node sample, a urine sample, a peritoneal fluid sample, a lavage sample, an esophageal brushing sample, a bladder or lung lavage sample, a cerebrospinal fluid sample, a brain fluid sample, a ductal aspirate sample, a breast secretion sample, a pleural effusion sample, a fresh frozen tissue sample, a paraffin-embedded tissue sample, In other embodiments, the sample is an extract from or a processed sample produced from any of a peripheral blood sample, a tumor tissue or suspected tumor tissue, a thin film cytology sample, a fine needle aspirate sample, a bone marrow sample, a urine sample, a peritoneal fluid sample, a lavage sample, an esophageal brushing sample, a bladder or lung lavage sample, a cerebrospinal fluid sample, a brain fluid sample, a ductal aspirate sample, a breast secretion sample, a pleural effusion sample, a fresh frozen tissue sample, or a paraffin-embedded tissue sample.

[0067] In some embodiments provided herein, the sample comprises cells from a cell line. In one embodiment, the cell line is a cell line such as Vero cells, CHO cells, MDCK cells, 293 T cells, HEK293T cells, Expi293F cells, BHK cells, HEK 293 cells, NS0 cells, PER.C6 cells, CRL7O3O cells, HsS78Bst cells, HeLa cells, NIH 3T3 cells, or other cell lines.

[0068] In some embodiments of any one of the above or below embodiments, the sample comprises cells from an immortalized cell line.

[0069] As used herein, an "immortalized cell line" describes a population of cells that can avoid normal cellular senescence and instead continue to undergo division due to a mutation(s). Thus, cells from an immortalized cell line can be grown for long periods of time in vitro. The mutations required for immortality can occur naturally or can be purposely induced for experimental purposes. Immortalized cells can be neoplastic / cancerous cells that do not stop dividing or cells that have been artificially engineered to grow indefinitely and therefore can be cultured for several generations. Without limitation, immortalized cell lines can include, for example, airway endothelial cells, aortic endothelial cells, Barrett's esophageal epithelial cells, bronchial epithelial cells, respiratory epithelial cells, chondrocyte fibroblasts, dermal microvascular endothelial cells (including TIME cells), endometrial fibroblasts, foreskin keratinocytes, pulmonary endothelial cells, mammary epithelial cells, mesenchymal stem cells, NTAP Schwann cells, pancreatic ductal cells, prostate cells, renal epithelial cells, retinal pigment epithelial cells, and skin fibroblasts.

[0070] In some embodiments of the above or below embodiments, methods are provided in which the sample comprises cells from an immortalized cell line. The immortalized cell line may be commercially available for use as described herein. A variety of immortalized cell lines are well known and commercially available (ATCC®, Manassas, VA).

[0071] As used herein, the term "specificity" generally refers to the ability of an assay to detect an antibody that reacts with a specific protein.For example, specificity can refer to a proportional detection response with a specific analyte, while the response to non-specific proteins should be less than LOD.Proportional response can be evaluated against a correlation coefficient R value of 0.98 or more.When used in connection with the method provided herein for detecting a target antigen, specificity refers to the ability to detect an antigen that reacts with a specific protein.

[0072] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is preferably a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans), most preferably a human. In one embodiment, the subject is a mammal, preferably a human. In some embodiments of the methods and kits provided herein, the patient has a disease or condition, or cancer. In other embodiments of the methods and kits provided herein, the patient is a patient undergoing cancer therapy. In yet other embodiments of the methods and kits provided herein, the patient is a pregnant woman or an infant (e.g., 0 to about 36 months of age).

[0073] As used herein, the terms "tag" and "label" are used interchangeably and refer to any type of moiety attached to an antibody or antigen-binding fragment thereof, or other polypeptide used in the methods provided herein. The term "detectable" or "detection" with respect to an antibody or tag refers to any antibody or tag that can be visualized, or the presence of the antibody or tag can be otherwise determined and / or measured (e.g., by quantification). Non-limiting examples of detectable tags include fluorescent or other chemiluminescent tags, and tags that can be amplified and quantified using PCR. In certain embodiments, the secondary antibody used in the methods provided herein is a biotinylated secondary antibody used in combination with labeled streptavidin.

[0074] As used herein, the term "therapy" refers to any protocol, method, and / or agent that may be used in the prevention, management, treatment, and / or amelioration of a disease (or symptoms associated therewith) or cancer. In certain embodiments, the terms "therapies" and "therapy" refer to biological, supportive, and / or other therapies useful in the prevention, management, treatment, and / or amelioration of a disease or cancer known to one of skill in the art, such as a medical practitioner.

[0075] As used herein, the term "tissue" refers to tissue obtained from a mammal, such as a human. For example, the tissue may be from a biopsy sample, surgically removed tissue, or a post-mortem collection. Additionally, the tissue may be homogenized and extracted to isolate enzymes or antibodies from the tissue.

[0076] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or remission of the progression, severity, and / or duration of a disease (or its associated symptoms) or cancer resulting from the administration of one or more therapies.

[0077] As used herein, the term "variant" refers to a polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the coding region relative to the original polypeptide coding domain. Variants retain the biological activity of the naturally occurring polypeptide.

[0078] As used herein, the term "in situ hybridization" or "ISH" refers to a technique for localizing and visualizing specific target nucleic acids while preserving the morphology of the source sample.

[0079] As used herein, the term "immunohistochemistry" or "IHC" refers to a technique for detecting a protein of interest in a source sample utilizing antibodies while preserving the morphology of the source sample. Immunofluorescence (IF) refers to fluorescent labeling and is therefore also encompassed by the term IHC.

[0080] As used herein, the term "crosslinking" refers to the process of linking two or more molecules together. A "crosslinker" or equivalent refers to an agent that contains two or more chemically reactive termini that attach themselves to functional groups found on proteins and other molecules. Specifically, when the crosslinker is formaldehyde or its equivalent, the nucleophilic group on an amino acid or nucleic acid base forms a covalent bond with the formaldehyde, which is often stabilized in a second step with another functional group on another molecule, resulting in the formation of a methylene bridge. When the crosslinker is an oxidizing agent, it can react with the side chains of proteins and other biomolecules, allowing the formation of crosslinks that stabilize tissue structures.

[0081] As used herein, the term "fixation" or "fixing", when performed with respect to fixation of a sample in an IHC process, refers to a procedure to protect the sample from decay, for example by autolysis or putrefaction, which may terminate any ongoing biochemical reactions and also increase the mechanical strength or stability of the processed tissue.

[0082] As used herein, the term "detecting" generally refers to any form of measurement, including determining whether an element is present or not. The term includes quantitative and / or qualitative determinations.

[0083] 5.2 IHC Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) tissues is a critical step in R&D therapeutic campaigns by identifying cells expressing target proteins of interest and predicting potential toxicity. A robust IHC assay relies on a suitable primary antibody that reliably recognizes the target with optimal specificity and sensitivity. FFPE tissues often present conformationally altered overfixed proteins, which makes repurposing of antibodies validated in non-IHC assays highly uncertain. If crosslinking agents such as formalin are used during the tissue preparation and preservation process, formalin fixation can mask epitopes and result in reduced immunoreactivity (see Arnold et al., Biotech Histochem 71:224-230 (1996)). Formalin fixation is a time-dependent process in which increasing fixation times result in successive formaldehyde group binding to proteins up to an equilibrium point (see Fox et al., J Histochem Cytochem 33:845-853 (1985)). Studies have shown that formalin fixation, especially if prolonged, results in reduced antigenicity (see Battifora and Kopinski, J Histochem Cytochem 34:1095-1100 (1986)), which limits the use of formalin-fixed tissues for diagnostic IHC (see Ramos-Vara, Vet Pathol 42:405-426 (2005); Webster et al., J Histochem Cytochem. 57(8):753-761 (2009)). The generation of new IHC antibodies requires screening many candidates against relevant controls when suitable reagents are not commercially available.

[0084] The method provided herein overcomes the above-mentioned challenges by modifying the conventional protocol. A method is provided herein that allows for screening of FFPE IHC quality antibodies using fixed cell lines (mimicking FFPE tissues) while maintaining them as single cell suspensions suitable for cell-based ELISA assays. This protocol allows for high-throughput testing of many antibodies and conditions that would be impossible to do in a timely manner using conventional controls mounted on glass slides.

[0085] 5.3 Methods for Enhancing Molecular Detection and Screening In one aspect, provided herein is a method of detecting a molecule in a sample, comprising obtaining cells from the sample, treating the cells with a fixative, then embedding the fixed cells in paraffin, subsequently deparaffinizing and suspending the cells to obtain a single cell suspension, then contacting the suspended cells with a first detection agent, then contacting the cells bound to the first detection agent with a second detection agent, and detecting the presence of the second detection agent bound to the cells of the sample.

[0086] In a preferred embodiment, the sample is obtained from the subject prior to performing the steps of the method.

[0087] In some embodiments, the first and / or second detection agent comprises an antibody or antigen-binding fragment thereof that binds to at least one molecule of the suspended cells.

[0088] In some embodiments, unbound cells are removed after contacting the suspended cells with a first detection agent.

[0089] In some embodiments, the second detection agent is an antibody or a fragment thereof.

[0090] In some embodiments, cells bound to a first detection agent are contacted with a second detection agent and then unbound second detection agent is removed.

[0091] In one embodiment of the methods provided herein, detection of an above background amount of a second detection agent bound to the sample indicates the presence of at least one molecule in the sample.

[0092] In some embodiments, the cells are cultured in a collodion bag and pelleted before being treated with a fixative. In one embodiment, the collodion bag is produced by coating the inner surface of a glass conical tube with a collodion solution. In one aspect, the collodion solution is first poured into a glass test tube, the solution is left for 1 hour, the collodion is poured back while swirling the tube, the tube is inverted to dry for 1 hour, and then the tube is filled with tap water, covered with parafilm, and stored upright in a refrigerator at 4°C until use. In one aspect, the collodion bag is prepared as described in Wilgenbusch et al., 2020 (Journal of the American Society of Cytopathology; 9, 20-25).

[0093] In some embodiments, the molecule is a nucleic acid or a protein. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is DNA.

[0094] In one aspect, provided herein is a method of detecting RNA in a sample, comprising obtaining cells from the sample, treating the cells with a fixative, then paraffin-embedding the fixed cells, subsequently deparaffinizing and suspending the cells to obtain a single cell suspension, then contacting the suspended cells with a first detection agent, then contacting the cells bound to the first detection agent with a second detection agent, and detecting the presence of the second detection agent bound to the cells of the sample.

[0095] In one aspect, provided herein is a method of detecting DNA in a sample, comprising obtaining cells from the sample, treating the cells with a fixative, then embedding the fixed cells in paraffin, subsequently deparaffinizing and suspending the cells to obtain a single cell suspension, then contacting the suspended cells with a first detection agent, then contacting the cells bound to the first detection agent with a second detection agent, and detecting the presence of the second detection agent bound to the cells of the sample.

[0096] In one aspect, provided herein is a method of detecting a protein in a sample, comprising obtaining cells from the sample, treating the cells with a fixative, then paraffin-embedding the fixed cells, subsequently deparaffinizing and suspending the cells to obtain a single cell suspension, then contacting the suspended cells with a first detection agent, then contacting the cells bound to the first detection agent with a second detection agent, and detecting the presence of the second detection agent bound to the cells of the sample.

[0097] In some embodiments, the fixative is selected from the group including formaldehyde, paraformaldehyde, glutaraldehyde, or neutral buffered formalin. In some embodiments, the fixative is formaldehyde. In some embodiments, the fixative is paraformaldehyde. In some embodiments, the fixative is glutaraldehyde. In some embodiments, the fixative is neutral buffered formalin.

[0098] In some embodiments, the methods provided herein include treating the sample with a fixative that is a mixture of fixatives. In some embodiments, the fixative is a mixed solution of two or more fixatives selected from the list of formaldehyde, glutaraldehyde, paraformaldehyde, neutral buffered formalin, acrolein, osmium tetroxide, permanganate fixative, dichromate fixative, and chromate. In one embodiment, the fixative is Bouin's fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In one embodiment, the fixative is a mixture of formaldehyde and glutaraldehyde. In one embodiment, the fixative is FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In one embodiment, the fixative is periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaO4. In one embodiment, the fixative is phosphate buffered formalin (PBF). In one embodiment, the fixative is formal-calcium, which is a solution of formaldehyde and calcium chloride. In one embodiment, the fixative is formal saline, which is a solution of formaldehyde and sodium chloride. In one embodiment, the fixative is zinc formalin, which is a solution of formaldehyde and zinc sulfate. In one embodiment, the fixative is Helly's fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercuric chloride. In one embodiment, the fixative is Hollande's fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid. In one embodiment, the fixative is Gendl's solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid. In one embodiment, the fixative is alcoholic formalin, which is a solution of formaldehyde, ethanol, and calcium acetate. In one embodiment, the fixative is formol acetate, which is a solution of formaldehyde, glacial acetic acid, and ethanol.In one embodiment, the fixative is a mixture of fixatives, where at least one fixative of the mixture is formaldehyde, neutral buffered formalin, or glutaraldehyde. In one embodiment, the fixative is a fixative used separately or sequentially, but not simultaneously, where at least one fixative is formaldehyde, neutral buffered formalin, or glutaraldehyde.

[0099] In some embodiments, the step of treating the cells with a fixative lasts for about 30 minutes to 48 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 60 minutes to 36 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 2 hours to 24 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 5 hours to 20 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 10 hours to 15 hours. In some embodiments, the step of treating the cells with the fixative lasts for about 30 minutes to 60 minutes, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 7 hours, 7 hours to 8 hours, 8 hours to 9 hours, 9 hours to 10 hours, 10 hours to 11 hours, 11 hours to 12 hours, 12 hours to 13 hours, 13 hours to 14 hours, 14 hours to 15 hours, 15 hours to 16 hours, 16 hours to 17 hours, 17 hours to 18 hours, 18 hours to 19 hours, 19 hours to 20 hours, 20 hours to 21 hours, 21 hours to 22 hours, 22 hours to 23 hours, 23 hours to 24 hours, 24 hours to 36 hours, or 36 hours to 48 hours. In some embodiments, the step of treating the cells with the fixative lasts for about 30 minutes to 60 minutes. In some embodiments, the step of treating the cells with a fixative lasts for about 1-2 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 2-3 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 3-4 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 4-5 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 5-6 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 6-7 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 7-8 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 8-9 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 9-10 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 10-11 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 11-12 hours.In some embodiments, the step of treating the cells with a fixative lasts for about 12 to 13 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 13 to 14 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 14 to 15 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 15 to 16 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 16 to 17 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 17 to 18 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 18 to 19 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 19 to 20 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 20 to 21 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 21 to 22 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 22 to 23 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 23 hours to about 24 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 24 hours to about 36 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 36 hours to about 48 hours.

[0100] In some embodiments, the step of treating the cells with the fixative lasts for about 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 36 hours, or 48 hours. In some embodiments, the step of treating the cells with the fixative lasts for about 30 minutes. In some embodiments, the step of treating the cells with the fixative lasts for about 60 minutes. In some embodiments, the step of treating the cells with the fixative lasts for about 2 hours. In some embodiments, the step of treating the cells with the fixative lasts for about 3 hours. In some embodiments, the step of treating the cells with the fixative lasts for about 4 hours. In some embodiments, the step of treating the cells with the fixative lasts for about 5 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 6 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 7 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 8 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 9 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 10 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 11 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 12 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 13 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 14 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 15 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 16 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 17 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 18 hours. In some embodiments, the step of treating the cells with the fixative lasts for about 19 hours. In some embodiments, the step of treating the cells with the fixative lasts for about 20 hours. In some embodiments, the step of treating the cells with the fixative lasts for about 21 hours.In some embodiments, the step of treating the cells with a fixative lasts for about 22 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 23 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 24 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 25 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 26 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 27 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 28 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 29 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 30 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 36 hours. In some embodiments, the step of treating the cells with a fixative lasts for about 48 hours.

[0101] In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 0° C. and 100° C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 1° C. and 90° C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 2° C. and 80° C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 3° C. and 70° C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 4° C. and 60° C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 0° C. and 10° C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 10° C. and 20° C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 20° C. and 30° C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 30° C. and 40° C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 40°C and 50°C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 50°C and 60°C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 60°C and 70°C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 70°C and 80°C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 80°C and 90°C. In some embodiments, the methods provided herein include treating the sample with a fixative at a temperature between 90°C and 100°C.

[0102] In some embodiments, treating the cells with a fixative is performed at 4° C., room temperature, 40° C., or 60° C. In some embodiments, treating the cells with a fixative is performed at 4° C. In some embodiments, treating the cells with a fixative is performed at room temperature. In some embodiments, treating the cells with a fixative is performed at 40° C. In some embodiments, treating the cells with a fixative is performed at 60° C.

[0103] In some embodiments, the methods provided herein include treating the sample with 1%-20% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 1% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 5% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 8% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 12% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 15% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 20% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 1%-5% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 5%-10% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 10%-15% neutral buffered formalin. In some embodiments, the methods provided herein include treating the sample with 15%-20% neutral buffered formalin.

[0104] In some embodiments, the methods provided herein include treating the sample with 1%-20% neutral buffered formalin at a temperature between 0°C and 100°C. In some embodiments, the methods provided herein include treating the sample with 1%-20% neutral buffered formalin at a temperature between 1°C and 90°C. In some embodiments, the methods provided herein include treating the sample with 1%-20% neutral buffered formalin at a temperature between 2°C and 80°C. In some embodiments, the methods provided herein include treating the sample with 1%-20% neutral buffered formalin at a temperature between 3°C and 70°C. In some embodiments, the methods provided herein include treating the sample with 1%-20% neutral buffered formalin at a temperature between 4°C and 60°C.

[0105] In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 0° C. and 20° C. In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 20° C. and 40° C. In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 40° C. and 60° C. In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 60° C. and 80° C. In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 80° C. and 100° C.

[0106] In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 0° C. and 100° C. In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 1° C. and 90° C. In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 2° C. and 80° C. In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 3° C. and 70° C. In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin at a temperature between 4° C. and 60° C.

[0107] In some embodiments, the methods provided herein include treating the sample with 10% neutral buffered formalin ("NBF") at a temperature of about 1° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 2° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 3° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 4° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 5° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 6° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 7° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 8° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 9° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 10° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 11° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 12° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 13° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 14° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 15° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 16° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 17° C.In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 18° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 19° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 20° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 21° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 22° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 23° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 24° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 25° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 26° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 27° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 28° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 29° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 30° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 35° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 40° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 45° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 50° C. In some embodiments, the methods provided herein include treating the sample with 10% NBF at a temperature of about 55° C.In some embodiments, the methods provided herein include treating a sample with 10% NBF at a temperature of about 60° C. In some embodiments, the methods provided herein include treating a sample with 10% NBF at a temperature of about 65° C. In some embodiments, the methods provided herein include treating a sample with 10% NBF at a temperature of about 70° C. In some embodiments, the methods provided herein include treating a sample with 10% NBF at a temperature of about 75° C. In some embodiments, the methods provided herein include treating a sample with 10% NBF at a temperature of about 80° C. In some embodiments, the methods provided herein include treating a sample with 10% NBF at a temperature of about 85° C. In some embodiments, the methods provided herein include treating a sample with 10% NBF at a temperature of about 90° C. In some embodiments, the methods provided herein include treating a sample with 10% NBF at a temperature of about 95° C. In some embodiments, the methods provided herein include treating a sample with 10% NBF at a temperature of about 100° C.

[0108] In some embodiments, the methods provided herein include treating the biological sample with 1%-20% NBF for 0.1 hours to 48 hours. In some embodiments, the methods provided herein include treating the biological sample with 1%-20% NBF for 0.1 hours to 36 hours. In some embodiments, the methods provided herein include treating the biological sample with 1%-20% NBF for 0.1 hours to 24 hours. In some embodiments, the methods provided herein include treating the biological sample with 1%-20% NBF for 0.2 hours to 22 hours. In some embodiments, the methods provided herein include treating the biological sample with 1%-20% NBF for 0.25 hours to 20 hours. In some embodiments, the methods provided herein include treating the biological sample with 1%-20% NBF for 0.25 hours to 18 hours.

[0109] In some embodiments, the methods provided herein include treating the biological sample with 10% NBF for 0.1 hours to 48 hours. In some embodiments, the methods provided herein include treating the biological sample with 10% NBF for 0.1 hours to 36 hours. In some embodiments, the methods provided herein include treating the biological sample with 10% NBF for 0.1 hours to 24 hours. In some embodiments, the methods provided herein include treating the biological sample with 10% NBF for 0.2 hours to 22 hours. In some embodiments, the methods provided herein include treating the biological sample with 10% NBF for 0.25 hours to 20 hours. In some embodiments, the methods provided herein include treating the biological sample with 10% NBF for 0.25 hours to 18 hours.

[0110] In some embodiments, treating the cells with a fixative is performed at room temperature for 24 hours. In some embodiments, treating the cells with 10% NBF is performed at room temperature for 24 hours.

[0111] In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 4° C. for more than 10 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 4° C. for more than 5 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 4° C. for more than 1 hour. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 4° C. for about 5 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 4° C. for about 6 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 4° C. for about 7 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 4° C. for about 8 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 4° C. for about 9 hours. In some specific embodiments, the methods provided herein comprise treating the biological sample with a fixative for about 10 hours at a temperature of 4° C. In some specific embodiments, the methods provided herein comprise treating the biological sample with a fixative for about 11 hours at a temperature of 4° C. In some specific embodiments, the methods provided herein comprise treating the biological sample with a fixative for about 12 hours at a temperature of 4° C.

[0112] In some particular embodiments, the methods provided herein include treating the biological sample with a fixative at room temperature for less than 6 hours. In some particular embodiments, the methods provided herein include treating the biological sample with a fixative at room temperature for less than 3 hours. In some particular embodiments, the methods provided herein include treating the biological sample with a fixative at room temperature for less than 1 hour. In some particular embodiments, the methods provided herein include treating the biological sample with a fixative at room temperature for less than 0.5 hours. In some particular embodiments, the methods provided herein include treating the biological sample with a fixative at room temperature for about 0.1 hours. In some particular embodiments, the methods provided herein include treating the biological sample with a fixative at room temperature for about 0.15 hours. In some particular embodiments, the methods provided herein include treating the biological sample with a fixative at room temperature for about 0.2 hours. In some particular embodiments, the methods provided herein include treating the biological sample with a fixative at room temperature for about 0.25 hours. In some particular embodiments, the methods provided herein include treating the biological sample with a fixative at room temperature for about 0.3 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.35 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.4 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.45 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.5 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.55 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.6 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.65 hours.In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.7 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.75 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.8 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.85 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 0.9 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 1 hour. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 1.5 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 2 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 2.5 hours. In some specific embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 3 hours. In some specific embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 3.5 hours. In some specific embodiments, the methods provided herein comprise treating the biological sample with a fixative at room temperature for about 4 hours.

[0113] In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 40° C. for less than 6 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 40° C. for less than 3 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 40° C. for less than 1 hour. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 40° C. for less than 0.5 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 40° C. for about 0.1 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 40° C. for about 0.25 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 40° C. for about 0.5 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 40° C. for about 0.75 hours. In some specific embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 40° C. for about 1 hour.

[0114] In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 60° C. for less than 6 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 60° C. for less than 3 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 60° C. for less than 1 hour. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 60° C. for less than 0.5 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 60° C. for about 0.1 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 60° C. for about 0.25 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 60° C. for about 0.5 hours. In some particular embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 60° C. for about 0.75 hours. In some specific embodiments, the methods provided herein comprise treating the biological sample with a fixative at a temperature of 60° C. for about 1 hour.

[0115] In one aspect of the methods provided herein, paraffin embedding of the fixed cells comprises contacting the cells with ethanol, then contacting the cells with xylene, and incubating the cells with paraffin.

[0116] In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 1% to 100% ethanol. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 5% to 95% ethanol. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 10% to 90% ethanol. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 15% to 85% ethanol. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 20% to 80% ethanol. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 25% to 75% ethanol. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 30% to 70% ethanol. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 35% to 65% ethanol. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 40% to 60% ethanol.

[0117] In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.1 hours to 2 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.5 hours to 1.5 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.5 hours to 1 hour. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.1 hours to 0.5 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.5 hours to 1 hour. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 1 hour to 1.5 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 1.5 hours to 2 hours.

[0118] In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.1 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.2 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.3 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.4 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.5 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.6 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.7 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.8 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 0.9 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 1 hour. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 1.5 hours. In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with ethanol for 2 hours.

[0119] In some embodiments, contacting the cells with ethanol comprises contacting the cells with 65%-75% ethanol, then contacting the cells with 75%-85% ethanol in water, then contacting the cells with 90%-100% ethanol in water for 30 minutes. In some embodiments, contacting the cells with ethanol comprises multiple concentrations of ethanol at multiple time intervals.

[0120] In some embodiments, the step of contacting the cells with ethanol comprises contacting the cells with 70% ethanol in water for 30 minutes, then contacting the cells with 80% ethanol in water for 30 minutes, then contacting the cells with 95% ethanol in water for 30 minutes, and finally contacting the cells with 100% ethanol for 30 minutes.

[0121] In some embodiments, contacting the cells with xylene comprises two changes of xylene. In some embodiments, contacting the cells with xylene comprises three changes of xylene. In some embodiments, contacting the cells with xylene comprises four changes of xylene. In some embodiments, contacting the cells with xylene comprises five changes of xylene.

[0122] In some embodiments, contacting the cells with xylene comprises two changes of xylene for 5 minutes to 40 minutes each. In some embodiments, contacting the cells with xylene comprises two changes of xylene for 5 minutes each. In some embodiments, contacting the cells with xylene comprises two changes of xylene for 10 minutes each. In some embodiments, contacting the cells with xylene comprises two changes of xylene for 15 minutes each. In some embodiments, contacting the cells with xylene comprises two changes of xylene for 20 minutes each. In some embodiments, contacting the cells with xylene comprises two changes of xylene for 25 minutes each. In some embodiments, contacting the cells with xylene comprises two changes of xylene for 30 minutes each. In some embodiments, contacting the cells with xylene comprises two changes of xylene for 35 minutes each. In some embodiments, contacting the cells with xylene comprises two changes of xylene for 40 minutes each.

[0123] In some embodiments, contacting the cells with xylene comprises three changes of xylene for 5 minutes to 40 minutes each. In some embodiments, contacting the cells with xylene comprises three changes of xylene for 5 minutes each. In some embodiments, contacting the cells with xylene comprises three changes of xylene for 10 minutes each. In some embodiments, contacting the cells with xylene comprises three changes of xylene for 15 minutes each. In some embodiments, contacting the cells with xylene comprises three changes of xylene for 20 minutes each. In some embodiments, contacting the cells with xylene comprises three changes of xylene for 25 minutes each. In some embodiments, contacting the cells with xylene comprises three changes of xylene for 30 minutes each. In some embodiments, contacting the cells with xylene comprises three changes of xylene for 35 minutes each. In some embodiments, contacting the cells with xylene comprises three changes of xylene for 40 minutes each.

[0124] In some embodiments, contacting the cells with xylene comprises four changes of xylene for 5 minutes to 40 minutes each. In some embodiments, contacting the cells with xylene comprises four changes of xylene for 5 minutes each. In some embodiments, contacting the cells with xylene comprises four changes of xylene for 10 minutes each. In some embodiments, contacting the cells with xylene comprises four changes of xylene for 15 minutes each. In some embodiments, contacting the cells with xylene comprises four changes of xylene for 20 minutes each. In some embodiments, contacting the cells with xylene comprises four changes of xylene for 25 minutes each. In some embodiments, contacting the cells with xylene comprises four changes of xylene for 30 minutes each. In some embodiments, contacting the cells with xylene comprises four changes of xylene for 35 minutes each. In some embodiments, contacting the cells with xylene comprises four changes of xylene for 40 minutes each.

[0125] In some embodiments, contacting the cells with xylene comprises 5 changes of xylene for 5 minutes to 40 minutes each. In some embodiments, contacting the cells with xylene comprises 5 changes of xylene for 5 minutes each. In some embodiments, contacting the cells with xylene comprises 5 changes of xylene for 10 minutes each. In some embodiments, contacting the cells with xylene comprises 5 changes of xylene for 15 minutes each. In some embodiments, contacting the cells with xylene comprises 5 changes of xylene for 20 minutes each. In some embodiments, contacting the cells with xylene comprises 5 changes of xylene for 25 minutes each. In some embodiments, contacting the cells with xylene comprises 5 changes of xylene for 30 minutes each. In some embodiments, contacting the cells with xylene comprises 5 changes of xylene for 35 minutes each. In some embodiments, contacting the cells with xylene comprises 5 changes of xylene for 40 minutes each.

[0126] In some embodiments, incubating the cells with paraffin comprises two changes of paraffin. In some embodiments, incubating the cells with paraffin comprises three changes of paraffin. In some embodiments, incubating the cells with paraffin comprises four changes of paraffin. In some embodiments, incubating the cells with paraffin comprises five changes of paraffin.

[0127] In some embodiments, incubating the cells with paraffin includes two changes of paraffin for 5 minutes to 40 minutes each. In some embodiments, incubating the cells with paraffin includes two changes of paraffin for 5 minutes each. In some embodiments, incubating the cells with paraffin includes two changes of paraffin for 10 minutes each. In some embodiments, incubating the cells with paraffin includes two changes of paraffin for 15 minutes each. In some embodiments, incubating the cells with paraffin includes two changes of paraffin for 20 minutes each. In some embodiments, incubating the cells with paraffin includes two changes of paraffin for 25 minutes each. In some embodiments, incubating the cells with paraffin includes two changes of paraffin for 30 minutes each. In some embodiments, incubating the cells with paraffin includes two changes of paraffin for 35 minutes each. In some embodiments, incubating the cells with paraffin includes two changes of paraffin for 40 minutes each.

[0128] In some embodiments, incubating the cells with paraffin includes three changes of paraffin for 5 minutes to 40 minutes each. In some embodiments, incubating the cells with paraffin includes three changes of paraffin for 5 minutes each. In some embodiments, incubating the cells with paraffin includes three changes of paraffin for 10 minutes each. In some embodiments, incubating the cells with paraffin includes three changes of paraffin for 15 minutes each. In some embodiments, incubating the cells with paraffin includes three changes of paraffin for 20 minutes each. In some embodiments, incubating the cells with paraffin includes three changes of paraffin for 25 minutes each. In some embodiments, incubating the cells with paraffin includes three changes of paraffin for 30 minutes each. In some embodiments, incubating the cells with paraffin includes three changes of paraffin for 35 minutes each. In some embodiments, incubating the cells with paraffin includes three changes of paraffin for 40 minutes each.

[0129] In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 5 to 40 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 5 to 10 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 10 to 15 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 15 to 20 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 20 to 25 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 25 to 30 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 30 to 35 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 35 to 40 minutes each.

[0130] In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 5 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 10 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 15 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 20 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 25 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 30 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 35 minutes each. In some embodiments, incubating the cells with paraffin includes four changes of paraffin for 40 minutes each.

[0131] In some embodiments, incubating the cells with paraffin includes 5 changes of paraffin for 5 minutes to 40 minutes each. In some embodiments, incubating the cells with paraffin includes 5 changes of paraffin for 5 minutes each. In some embodiments, incubating the cells with paraffin includes 5 changes of paraffin for 10 minutes each. In some embodiments, incubating the cells with paraffin includes 5 changes of paraffin for 15 minutes each. In some embodiments, incubating the cells with paraffin includes 5 changes of paraffin for 20 minutes each. In some embodiments, incubating the cells with paraffin includes 5 changes of paraffin for 25 minutes each. In some embodiments, incubating the cells with paraffin includes 5 changes of paraffin for 30 minutes each. In some embodiments, incubating the cells with paraffin includes 5 changes of paraffin for 35 minutes each. In some embodiments, incubating the cells with paraffin includes 5 changes of paraffin for 40 minutes each.

[0132] In some embodiments, the step of incubating the cells with paraffin is performed at 5° C. to 100° C. In some embodiments, the step of incubating the cells with paraffin is performed at 10° C. to 90° C. In some embodiments, the step of incubating the cells with paraffin is performed at 20° C. to 80° C. In some embodiments, the step of incubating the cells with paraffin is performed at 30° C. to 70° C. In some embodiments, the step of incubating the cells with paraffin is performed at 40° C. to 60° C. In some embodiments, the step of incubating the cells with paraffin is performed at 0° C. to 10° C. In some embodiments, the step of incubating the cells with paraffin is performed at 10° C. to 20° C. In some embodiments, the step of incubating the cells with paraffin is performed at 20° C. to 30° C. In some embodiments, the step of incubating the cells with paraffin is performed at 30° C. to 40° C. In some embodiments, the step of incubating the cells with paraffin is performed at 40° C. to 50° C. In some embodiments, the step of incubating the cells with paraffin is performed at 50° C. to 60° C. In some embodiments, the step of incubating the cells with paraffin is performed at 60°C to 70°C. In some embodiments, the step of incubating the cells with paraffin is performed at 70°C to 80°C. In some embodiments, the step of incubating the cells with paraffin is performed at 80°C to 90°C. In some embodiments, the step of incubating the cells with paraffin is performed at 90°C to 100°C.

[0133] In some embodiments, the step of incubating the cells with paraffin is performed at 5° C. In some embodiments, the step of incubating the cells with paraffin is performed at 10° C. In some embodiments, the step of incubating the cells with paraffin is performed at 15° C. In some embodiments, the step of incubating the cells with paraffin is performed at 20° C. In some embodiments, the step of incubating the cells with paraffin is performed at 25° C. In some embodiments, the step of incubating the cells with paraffin is performed at 30° C. In some embodiments, the step of incubating the cells with paraffin is performed at 35° C. In some embodiments, the step of incubating the cells with paraffin is performed at 40° C. In some embodiments, the step of incubating the cells with paraffin is performed at 45° C. In some embodiments, the step of incubating the cells with paraffin is performed at 50° C. In some embodiments, the step of incubating the cells with paraffin is performed at 55° C. In some embodiments, the step of incubating the cells with paraffin is performed at 60° C. In some embodiments, the step of incubating the cells with paraffin is performed at 65° C. In some embodiments, the step of incubating the cells with paraffin is performed at 70° C. In some embodiments, the step of incubating the cells with paraffin is performed at 75° C. In some embodiments, the step of incubating the cells with paraffin is performed at 80° C. In some embodiments, the step of incubating the cells with paraffin is performed at 85° C. In some embodiments, the step of incubating the cells with paraffin is performed at 90° C. In some embodiments, the step of incubating the cells with paraffin is performed at 95° C. In some embodiments, the step of incubating the cells with paraffin is performed at 100° C.

[0134] In some embodiments, the cells are deparaffinized by contacting the cells with xylene. In some embodiments, the cells are contacted with xylene for about 5 to 120 minutes. In some embodiments, the cells are contacted with xylene for about 5 to 10 minutes, 10 to 15 minutes, 15 to 20 minutes, 20 to 30 minutes, 30 to 60 minutes, 60 to 90 minutes, or 90 to 120 minutes. In some embodiments, the cells are contacted with xylene for about 5 minutes. In some embodiments, the cells are contacted with xylene for about 10 minutes. In some embodiments, the cells are contacted with xylene for about 15 minutes. In some embodiments, the cells are contacted with xylene for about 20 minutes. In some embodiments, the cells are contacted with xylene for about 25 minutes. In some embodiments, the cells are contacted with xylene for about 30 minutes. In some embodiments, the cells are contacted with xylene for about 35 minutes. In some embodiments, the cells are contacted with xylene for about 40 minutes. In some embodiments, the cells are contacted with xylene for about 45 minutes. In some embodiments, the cells are contacted with xylene for about 50 minutes. In some embodiments, the cells are contacted with xylene for about 55 minutes. In some embodiments, the cells are contacted with xylene for about 60 minutes. In some embodiments, the cells are contacted with xylene for about 90 minutes. In some embodiments, the cells are contacted with xylene for about 120 minutes.

[0135] In some embodiments, the cells are further contacted with a continuous ethanol gradient, as described above.

[0136] In one aspect of the methods provided herein, the cells are resuspended in an antigen unmasking solution.

[0137] In some embodiments, the cells are further heated to between 60°C and 100°C. In some embodiments, the cells are further heated to between 60°C and 70°C. In some embodiments, the cells are further heated to between 70°C and 80°C. In some embodiments, the cells are further heated to between 80°C and 90°C. In some embodiments, the cells are further heated to between 90°C and 100°C. In some embodiments, the cells are further heated to 60°C. In some embodiments, the cells are further heated to 65°C. In some embodiments, the cells are further heated to 70°C. In some embodiments, the cells are further heated to 75°C. In some embodiments, the cells are further heated to 80°C. In some embodiments, the cells are further heated to 85°C. In some embodiments, the cells are further heated to 90°C. In some embodiments, the cells are further heated to 95°C. In some embodiments, the cells are further heated to 100°C.

[0138] In some embodiments, the cells are further heated at 60° C. for about 10 to 60 minutes. In some embodiments, the cells are further heated at 60° C. for about 10 to 20 minutes. In some embodiments, the cells are further heated at 60° C. for about 20 to 30 minutes. In some embodiments, the cells are further heated at 60° C. for about 30 to 40 minutes. In some embodiments, the cells are further heated at 60° C. for about 40 to 50 minutes. In some embodiments, the cells are further heated at 60° C. for about 50 to 60 minutes. In some embodiments, the cells are further heated at 60° C. for about 10 minutes. In some embodiments, the cells are further heated at 60° C. for about 20 minutes. In some embodiments, the cells are further heated at 60° C. for about 30 minutes. In some embodiments, the cells are further heated at 60° C. for about 40 minutes. In some embodiments, the cells are further heated at 60° C. for about 50 minutes. In some embodiments, the cells are further heated at 60° C. for about 60 minutes.

[0139] In some embodiments, the cells are further heated at 65° C. for about 10 to 60 minutes. In some embodiments, the cells are further heated at 65° C. for about 10 to 20 minutes. In some embodiments, the cells are further heated at 65° C. for about 20 to 30 minutes. In some embodiments, the cells are further heated at 65° C. for about 30 to 40 minutes. In some embodiments, the cells are further heated at 65° C. for about 40 to 50 minutes. In some embodiments, the cells are further heated at 65° C. for about 50 to 60 minutes. In some embodiments, the cells are further heated at 65° C. for about 10 minutes. In some embodiments, the cells are further heated at 65° C. for about 20 minutes. In some embodiments, the cells are further heated at 65° C. for about 30 minutes. In some embodiments, the cells are further heated at 65° C. for about 40 minutes. In some embodiments, the cells are further heated at 65° C. for about 50 minutes. In some embodiments, the cells are further heated at 65° C. for about 60 minutes.

[0140] In some embodiments, the cells are further heated at 70° C. for about 10 to 60 minutes. In some embodiments, the cells are further heated at 70° C. for about 10 to 20 minutes. In some embodiments, the cells are further heated at 70° C. for about 20 to 30 minutes. In some embodiments, the cells are further heated at 70° C. for about 30 to 40 minutes. In some embodiments, the cells are further heated at 70° C. for about 40 to 50 minutes. In some embodiments, the cells are further heated at 70° C. for about 50 to 60 minutes. In some embodiments, the cells are further heated at 70° C. for about 10 minutes. In some embodiments, the cells are further heated at 70° C. for about 20 minutes. In some embodiments, the cells are further heated at 70° C. for about 30 minutes. In some embodiments, the cells are further heated at 70° C. for about 40 minutes. In some embodiments, the cells are further heated at 70° C. for about 50 minutes. In some embodiments, the cells are further heated at 70° C. for about 60 minutes.

[0141] In some embodiments, the cells are further heated at 80° C. for about 10 to 60 minutes. In some embodiments, the cells are further heated at 80° C. for about 10 to 20 minutes. In some embodiments, the cells are further heated at 80° C. for about 20 to 30 minutes. In some embodiments, the cells are further heated at 80° C. for about 30 to 40 minutes. In some embodiments, the cells are further heated at 80° C. for about 40 to 50 minutes. In some embodiments, the cells are further heated at 80° C. for about 50 to 60 minutes. In some embodiments, the cells are further heated at 80° C. for about 10 minutes. In some embodiments, the cells are further heated at 80° C. for about 20 minutes. In some embodiments, the cells are further heated at 80° C. for about 30 minutes. In some embodiments, the cells are further heated at 80° C. for about 40 minutes. In some embodiments, the cells are further heated at 80° C. for about 50 minutes. In some embodiments, the cells are further heated at 80° C. for about 60 minutes.

[0142] In some embodiments, the cells are further heated at 85° C. for about 10 to 60 minutes. In some embodiments, the cells are further heated at 85° C. for about 10 to 20 minutes. In some embodiments, the cells are further heated at 85° C. for about 20 to 30 minutes. In some embodiments, the cells are further heated at 85° C. for about 30 to 40 minutes. In some embodiments, the cells are further heated at 85° C. for about 40 to 50 minutes. In some embodiments, the cells are further heated at 85° C. for about 50 to 60 minutes. In some embodiments, the cells are further heated at 85° C. for about 10 minutes. In some embodiments, the cells are further heated at 85° C. for about 20 minutes. In some embodiments, the cells are further heated at 85° C. for about 30 minutes. In some embodiments, the cells are further heated at 85° C. for about 40 minutes. In some embodiments, the cells are further heated at 85° C. for about 50 minutes. In some embodiments, the cells are further heated at 85° C. for about 60 minutes.

[0143] In some embodiments, the cells are further heated at 90° C. for about 10 to 60 minutes. In some embodiments, the cells are further heated at 90° C. for about 10 to 20 minutes. In some embodiments, the cells are further heated at 90° C. for about 20 to 30 minutes. In some embodiments, the cells are further heated at 90° C. for about 30 to 40 minutes. In some embodiments, the cells are further heated at 90° C. for about 40 to 50 minutes. In some embodiments, the cells are further heated at 90° C. for about 50 to 60 minutes. In some embodiments, the cells are further heated at 90° C. for about 10 minutes. In some embodiments, the cells are further heated at 90° C. for about 20 minutes. In some embodiments, the cells are further heated at 90° C. for about 30 minutes. In some embodiments, the cells are further heated at 90° C. for about 40 minutes. In some embodiments, the cells are further heated at 90° C. for about 50 minutes. In some embodiments, the cells are further heated at 90° C. for about 60 minutes.

[0144] In some embodiments, the cells are further heated at 95° C. for about 10 to 60 minutes. In some embodiments, the cells are further heated at 95° C. for about 10 to 20 minutes. In some embodiments, the cells are further heated at 95° C. for about 20 to 30 minutes. In some embodiments, the cells are further heated at 95° C. for about 30 to 40 minutes. In some embodiments, the cells are further heated at 95° C. for about 40 to 50 minutes. In some embodiments, the cells are further heated at 95° C. for about 50 to 60 minutes. In some embodiments, the cells are further heated at 95° C. for about 10 minutes. In some embodiments, the cells are further heated at 95° C. for about 20 minutes. In some embodiments, the cells are further heated at 95° C. for about 30 minutes. In some embodiments, the cells are further heated at 95° C. for about 40 minutes. In some embodiments, the cells are further heated at 95° C. for about 50 minutes. In some embodiments, the cells are further heated at 95° C. for about 60 minutes.

[0145] In some embodiments, the cells are further heated at 100° C. for about 10 to 60 minutes. In some embodiments, the cells are further heated at 100° C. for about 10 to 20 minutes. In some embodiments, the cells are further heated at 100° C. for about 20 to 30 minutes. In some embodiments, the cells are further heated at 100° C. for about 30 to 40 minutes. In some embodiments, the cells are further heated at 100° C. for about 40 to 50 minutes. In some embodiments, the cells are further heated at 100° C. for about 50 to 60 minutes. In some embodiments, the cells are further heated at 100° C. for about 10 minutes. In some embodiments, the cells are further heated at 100° C. for about 20 minutes. In some embodiments, the cells are further heated at 100° C. for about 30 minutes. In some embodiments, the cells are further heated at 100° C. for about 40 minutes. In some embodiments, the cells are further heated at 100° C. for about 50 minutes. In some embodiments, the cells are further heated to 100° C. for about 60 minutes.

[0146] In some embodiments, the cells are heated by microwave radiation, hi some embodiments, the cells are heated in a water bath.

[0147] In one aspect of the methods provided herein, the heating results in antigen retrieval.

[0148] In some embodiments, the first and / or second detection agent is an antibody or an antigen-binding fragment thereof. In some embodiments, the first and / or second detection agent is an RNA-based binder molecule. Any of a number of enzymes or non-enzyme labels can be used as detection agents, so long as the enzyme activity or non-enzyme label, respectively, can be detected. The enzyme thereby produces a detectable signal that can be used to detect the target molecule. Particularly useful detectable signals are chromogenic or fluorogenic signals. Such enzymes are well known to those skilled in the art and include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, and the like (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Other enzymes with well-known chromogenic or fluorogenic substrates include various peptidases, and chromogenic or fluorogenic peptide substrates can be used to detect proteolytic cleavage reactions. The use of chromogenic and fluorogenic substrates, including α- and β-galactosidase, β-glucuronidase, 6-phospho-β-D-galactoside 6-phosphogalactohydrolase, β-glucosidase, α-glucosidase, amylase, neuraminidase, esterase, lipase, and the like, is also well known in bacterial diagnostics (Manafi et al., Microbiol. Rev. 55:335-348 (1991)), and such enzymes with known chromogenic or fluorogenic substrates can be readily adapted for use in the methods provided herein.

[0149] A variety of chromogenic or fluorogenic substrates for producing a detectable signal are well known to those of skill in the art and are commercially available. Exemplary substrates that can be utilized to produce a detectable signal include 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), 4-chloronaphthol (4-CN(chloronaphthol)) (4-chloro-1-naphthol), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), o-phenylenediamine dihydrochloride (o-phenylenediamine dihydrochloride), and o-phenylenediamine dihydrochloride (o-phenylenediamine dihydrochloride). dihydrochloride (OPD), and 3-amino-9-ethylcarbazole (AEC); alkaline phosphatases include, but are not limited to, 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitroblue tetrazolium (NBT), Fast Red (Fast Red TR / AS-MX), and p-nitrophenyl phosphate (PNPP); β-galactosidases include, but are not limited to, 1-methyl-3-indolyl-β-D-galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-galactopyranoside; and β-glucosidases include, but are not limited to, 2-methoxy-4-(2-nitrovinyl)phenyl β-D-glucopyranoside.Exemplary fluorogenic substrates include 4-(trifluoromethyl)umbelliferyl phosphate for alkaline phosphatase; 4-methylumbelliferyl phosphate bis(2-amino-2-methyl-1,3-propanediol), 4-methylumbelliferyl phosphate bis(cyclohexylammonium) and 4-methylumbelliferyl phosphate for phosphatases; QuantaBlu™ and Quintolet for horseradish peroxidase; β-galactosidase; Exemplary enzymes and substrates for producing detectable signals include, but are not limited to, 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di(β-D-galactopyranoside), and naphthofluorescein di(β-D-galactopyranoside); β-glucosidases include 3-acetylumbelliferyl β-D-glucopyranoside and 4-methylumbelliferyl-β-D-glucopyranoside; and α-galactosidases include 4-methylumbelliferyl-α-D-galactopyranoside. Exemplary enzymes and substrates for producing detectable signals are also described, for example, in U.S. Patent Application Publication No. 2012 / 0100540. A variety of detectable enzyme substrates (including chromogenic or fluorogenic substrates) are well known and commercially available (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Invitrogen, Carlsbad CA; 42 Life Science; Biocare). Generally, the substrate is converted to a product that forms a precipitate that deposits at the site of the target nucleic acid. Other exemplary substrates include, but are not limited to, HRP-Green (42 Life Science), Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran Purple, Vina Green, Deep Space Black™, Warp Red™, Vulcan Fast Red (from Biocare (Concord CA; biocare.net / products / detection / chromogens)).

[0150] Exemplary rare earth metals and metal isotopes suitable as detectable labels include lanthanide (III) isotopes, e.g., 141 Pr, 142 Nd, 143 Nd, 144 Nd, 145 Nd, 146 Nd, 147 Sm, 148 Nd, 149 Sm, 150 Nd, 151 EU, 152 Sm, 153 EU, 154 Sm, 155 Gd, 156 Gd, 158 Gd, 159 Tb, 160 Gd, 161 Dy, 162 Dy, 163 Dy, 164 Dy, 165 Ho, 166 Er, 167 Er, 168 Er, 169 Tm, 170 Er, 171 Yb, 172 Yb, 173 Yb, 174 Yb, 175 Lu, and 176 Metal isotopes can be detected, for example, using time-of-flight mass spectrometry (TOF-MS) (e.g., Fluidigm Helios and Hyperion systems, fluidigm.com / systems; South San Francisco, Calif.).

[0151] Biotin-avidin (or biotin-streptavidin) is a well-known signal amplification system based on the fact that the two molecules have a very high affinity for each other, and one avidin / streptavidin molecule can bind four biotin molecules. Antibodies are widely used for signal amplification in immunohistochemistry. Tyramide signal amplification (TSA) is based on the deposition of many haptenized tyramide molecules by peroxidase activity. Tyramine is a phenolic compound. In the presence of small amounts of hydrogen peroxide, immobilized horseradish peroxidase (HRP) converts a labeled substrate into a short-lived, highly reactive intermediate. The activated substrate molecule then reacts very rapidly with and covalently binds to electron-rich moieties of proteins, such as tyrosine, at or near the site of the peroxidase binding site. In this way, many hapten molecules conjugated to tyramide can be introduced in situ to the hybridization site. The deposited tyramide-hapten molecules can then be visualized directly or indirectly. Such detection systems are described in more detail, for example, in U.S. Patent Application Publication No. 2012 / 0100540.

[0152] The embodiments described herein can utilize an enzyme to generate a detectable signal using an appropriate chromogenic or fluorogenic substrate. Alternatively, it is understood that the labeled probe can have a detectable label directly attached to the nucleic acid portion of the labeled probe. Exemplary detectable labels are well known to those of skill in the art and include, but are not limited to, chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Exemplary fluorophores useful as labels include rhodamine derivatives, such as tetramethylrhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, Texas Red (sulforhodamine 101), rhodamine 110, and derivatives thereof, such as tetramethylrhodamine-5-(or-6), Lissamine rhodamine B; 7-nitrobenz-2-oxa-1,3-diazole (NBD); fluorescein and its derivatives; naphthalenes, such as dansyl (5-dimethylaminonaphthalene-1-sulfonyl); 7-amino-4-methylcoumarin-3-acetic acid; coumarin derivatives such as 7-diethylamino-3-[(4'-(iodoacetyl)amino)phenyl]-4-methylcoumarin (AMCA), 7-diethylamino-3-[(4'-(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA), and Alexa fluor dyes (Molecular Probes); 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY™) and its derivatives (Molecular Probes; Eugene, OR); pyrene and sulfonated pyrenes such as Cascade Blue™ and its derivatives, including 8-methoxypyrene-1,3,6-trisulfonic acid; pyridyloxazole derivatives and dapoxyl derivatives (Molecular Probes); Lucifer Yellow (3,6-disulfonate-4-amino-naphthalimide) and its derivatives;CyDye™ Fluorescent Dye (Amersham / GE Healthcare Life Sciences; Piscataway NJ), ATTO 390, DyLight 395XL, ATTO 425, ATTO 465, ATTO 488, ATTO 490LS, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Examples of the dyes include, but are not limited to, Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, Cyan 500 NHS-Ester (ATTO-TECH, Siegen, Germany), etc. Exemplary chromophores include, but are not limited to, phenolphthalein, malachite green, nitroaromatic compounds such as nitrophenyl, diazo dyes, dabsyl (4-dimethylaminoazobenzene-4'-sulfonyl), etc.;

[0153] Well-known methods such as microscopy, cytometry (e.g., mass cytometry, cytometry by time of flight (CyTOF), flow cytometry), or spectroscopy can be utilized to visualize the chromogenic, fluorescent, or metal-detectable signals associated with each target nucleic acid. Generally, either a chromogenic or fluorogenic substrate, or a chromogenic or fluorogenic label, or a rare earth metal isotope, is utilized for a particular assay when different labels are used in the same assay, such that a single type of instrument can be used for detection of nucleic acid targets in the same sample.

[0154] In some embodiments, the methods provided herein include using a labeled primary antibody, thus eliminating the need to perform other IHC steps. In certain embodiments, the primary antibody is labeled with a chromogenic label. In certain embodiments, the primary antibody is labeled with a fluorescent label. In certain embodiments, the primary antibody is labeled with a polynucleotide(s). In certain embodiments, the primary antibody is labeled by the NHS (succinimidyl) ester method. In certain embodiments, the primary antibody is labeled by the isothiocyanate method. In certain embodiments, the primary antibody is labeled by the carbodiimide method. In certain embodiments, the primary antibody is labeled by the two-tag method (catalyst and its substrate). In certain embodiments, the primary antibody is labeled by the periodate method. Post-primary antibody crosslinking can be adapted for use with fluorescence-based detection, in combination with the Basescope™ signal amplification system (see Baker et al., Nature Communication 8:1998 (2017)), or in combination with other nucleic acid detection methods using similar protocols.

[0155] In one aspect of the methods provided herein, the sample comprises a body fluid or cells from a tissue. In some embodiments, the body fluid is blood, serum, or plasma. In one embodiment, the body fluid is blood. In one embodiment, the body fluid is serum. In one embodiment, the body fluid is plasma. In one embodiment, the sample is a tissue specimen or derived from a tissue specimen. In one aspect, the sample is a blood sample or derived from a blood sample. In one embodiment, the sample is a cytological sample or derived from a cytological sample. In one embodiment, the sample is cultured cells. In another embodiment, the sample is a sample containing exosomes. In some embodiments of any one of the above or below embodiments, the sample comprises an immortalized cell line.

[0156] Tissue specimens include, for example, tissue biopsy samples. Blood samples include, for example, blood samples taken for diagnostic purposes. In the case of blood samples, the blood can be analyzed directly, such as by blood smear, or the blood can be processed, such as by lysing red blood cells, isolating PBMCs or white blood cells, isolating target cells, etc., so that the cells in the sample analyzed by the method of the present disclosure are in or derived from the blood sample. Similarly, tissue specimens can be processed, such as by mincing the tissue specimen and treating it physically or enzymatically to break the tissue into individual cells or cell clusters. Additionally, cytological samples can be treated to isolate cells or break cell clusters, if desired. Thus, tissue, blood, and cytological samples can be obtained and processed using methods well known in the art. The method of the present disclosure can be used in diagnostic applications to identify the presence or absence of pathological cells based on the presence or absence of a nucleic acid target that is a biomarker indicative of pathology.

[0157] It will be understood by those skilled in the art that any of several suitable sample types can be used to detect target nucleic acids and target proteins using the methods provided herein. The samples for use in the methods provided herein are generally biological or tissue samples. Such samples can be obtained from biological subjects, including biological tissue or fluid origin samples collected from individuals or biological materials, such as biopsies, autopsies, or some other sources of forensic materials. Biological samples also include samples from areas of biological subjects that contain or are suspected of containing precancerous or cancerous cells or tissues, such as fine needle aspirates, blood samples, or tissue biopsies, including cytological specimens. Such samples can be, but are not limited to, organs, tissues, tissue fractions, cells, and / or exosomes isolated from organisms such as mammals. Exemplary biological samples include, but are not limited to, cell cultures containing cells, primary cell cultures, cell lines, tissues, organs, organoids, biological fluids, and the like. Further biological samples include, but are not limited to, skin samples, tissue biopsies including fine needle aspirates, cytological samples, feces, bodily fluids including blood and / or serum samples, saliva, semen, etc. Such samples may be used for medical or veterinary diagnostic purposes.

[0158] Collection of cytological samples for analysis by the methods provided herein is well known in the art (see, e.g., Dey, "Cytology Sample Procurement, Fixation and Processing" in Basic and Advanced Laboratory Techniques in Histopathology and Cytology pp.121-132, Springer, Singapore (2018); "Non-Gynecological Cytology Practice Guideline" American Society of Cytopathology, Adopted by the ASC executive board March 2, 2004).

[0159] For example, methods for processing samples for analysis of cervical tissue, including tissue biopsies and cytological samples, are well known in the art (e.g., Cecil Textbook of Medicine, Bennett and Plum, eds., 20th ed., W. B. Saunders, Philadelphia (1996); Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginner's Manual, Sellors and Sankaranarayanan, eds., International Agency for Research on Cancer, Lyon, France (2003); Kalaf and Cooper, J. Clin. Pathol. 60:449-455 (2007); Brown and Trimble, Best Pract. Res. Clin. Obstet. Gynaecol. 26:233-242 (2012); Waxman et al. See, e.g., et al., Obstet. Gynecol. 120:1465-1471 (2012); Cervical Cytology Practice Guidelines TOC, Approved by the American Society of Cytopathology (ASC) Executive Board, November 10, 2000).

[0160] In some embodiments of any one of the above or below embodiments, the cells comprise an immortalized cell line.

[0161] In another aspect, provided herein is a kit for carrying out the method of any one of the embodiments provided herein.In another aspect, provided herein is a sample of cells prepared by the method of any one of the embodiments provided herein.

[0162] The methods provided herein are valuable research tools as well as diagnostic tools. In some embodiments, the methods provided herein are used to map spatial organization in complex tissues. In some specific embodiments, the methods provided herein are used to identify cell types and new cell types. In some specific embodiments, the methods provided herein are used to identify cellular states. In other specific embodiments, the methods provided herein are used to identify cell types and new cell types in the tumor microenvironment. In some specific embodiments, the methods provided herein are used to identify cellular states in the tumor microenvironment.

[0163] In some embodiments, the methods provided herein may be used in an automated system. In some embodiments, the methods provided herein may be used in an automated process. In some embodiments, an automated system may be used for paraffin embedding of fixed cells. In some embodiments, an automated process may be used for paraffin embedding of fixed cells. In some embodiments, an automated system may be used for deparaffinization of cells. In some embodiments, an automated process may be used for deparaffinization of cells. In some embodiments of the various methods provided herein, each step may be independently either manual or automated. In some embodiments of the various methods provided herein, some steps are performed manually and other steps are automated.

[0164] In some embodiments, the methods provided herein are used to detect altered gene expression in diseased cells and tissues. In some particular embodiments, the methods provided herein are used to localize altered gene expression in specific cell types and understand tumor heterogeneity. In some particular embodiments, the methods provided herein are used to study tumor-immune cell interactions. In some embodiments, the methods provided herein are used to detect biomarkers for cancer diagnosis and prognosis. In some embodiments, the methods provided herein are used to detect therapeutic targets for cancer treatment. In some embodiments, the methods provided herein are used to facilitate validation of novel antibodies.

[0165] Embodiment The present invention provides the following non-limiting embodiments.

[0166] In one set of embodiments, the following is provided: 1. A method for detecting a molecule in a sample, comprising: a. obtaining cells from a sample; b. treating the cells with a fixative; c. embedding the fixed cells in paraffin; d. Deparaffinizing and suspending the cells to obtain a single cell suspension; e. contacting the suspended cells with a first detection agent that binds to at least one molecule of the suspended cells; f. contacting the cells bound to the first detection agent with a second detection agent; g. detecting the presence of a second detection agent bound to the cells of the sample; wherein detection of an above background amount of the second detection agent bound to the sample indicates the presence of at least one molecule in the sample. 2. The method of embodiment 1, wherein the molecule is a nucleic acid or a protein. 3. The method of embodiment 2, wherein the nucleic acid is RNA. 4. The method of embodiment 2, wherein the nucleic acid is DNA. 5. The method of any one of embodiments 1-4, wherein the method further comprises removing unbound cells after contacting the suspended cells with the first detection agent. 6. The method of any one of embodiments 1-5, wherein the method further comprises removing unbound second detection agent after contacting the cells bound to the first detection agent with a second detection agent. 7. The method of any one of the preceding claims, wherein the fixative is selected from the group comprising formaldehyde, paraformaldehyde, glutaraldehyde, or neutral buffered formalin. 8. The method of embodiment 7, wherein the fixative is neutral buffered formalin. 9. The method of embodiment 8, wherein the neutral buffered formalin is 10% neutral buffered formalin. 10. The method according to any one of the preceding embodiments, wherein the step of treating the cells with the fixative lasts for about 30 min to 60 min, 1 h to 2 h, 2 h to 3 h, 3 h to 4 h, 4 h to 5 h, 5 h to 6 h, 6 h to 7 h, 7 h to 8 h, 8 h to 9 h, 9 h to 10 h, 10 h to 11 h, 11 h to 12 h, 12 h to 13 h, 13 h to 14 h, 14 h to 15 h, 15 h to 16 h, 16 h to 17 h, 17 h to 18 h, 18 h to 19 h, 19 h to 20 h, 20 h to 21 h, 21 h to 22 h, 22 h to 23 h, 23 h to 24 h, 24 h to 36 h, or 36 h to 48 h. 11. The method according to any one of the preceding embodiments, wherein the step of treating the cells with a fixative is carried out at 4°C, room temperature, 40°C, or 60°C. 12. The method according to any one of the preceding claims, wherein the step of treating the cells with a fixative is carried out at room temperature for 24 hours. 13. Paraffin embedding of fixed cells a. contacting the cells with ethanol; b. contacting the cells with xylene; c. Incubating the cells with paraffin. 14. The step of contacting the cells with ethanol comprises: a. contacting the cells with 70% ethanol in water for 30 minutes; b. contacting the cells with 80% ethanol in water for 30 minutes; c. contacting the cells with 95% ethanol in water for 30 minutes; d. Contacting the cells with 100% ethanol for 30 minutes. 15. The method of embodiment 13 or 14, wherein the step of contacting the cells with xylene comprises three changes of xylene for 20 minutes each. 16. The method of any one of embodiments 13-15, wherein the step of incubating the cells with paraffin comprises four changes of paraffin for 20 minutes each. 17. The method of embodiment 16, wherein the step of incubating the cells with paraffin is carried out at 60°C. 18. The method of any one of embodiments 1 to 17, wherein the cells are deparaffinized by contacting the cells with xylene. 19. The method of embodiment 18, wherein the cells are contacted with xylene for about 5 minutes to 10 minutes, 10 minutes to 15 minutes, 15 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 60 minutes, 60 minutes to 90 minutes, or 90 minutes to 120 minutes. 20. The method of embodiment 18 or 19, wherein the cells are further contacted with a continuous ethanol gradient comprising contacting the cells with 70% ethanol in water for about 15 to about 30 minutes, contacting the cells with 95% ethanol in water for about 15 to about 30 minutes, and finally contacting the cells with 100% ethanol for about 15 to about 30 minutes. Embodiment 21. The method of any one of embodiments 1 to 20, wherein the cells are resuspended in an antigen retrieval solution. 22. The method of embodiment 21, wherein the cells are further heated to 95° C. for about 30 minutes. 23. The method of embodiment 22, wherein the cells are heated by microwave radiation. 24. The method of any one of embodiments 1-23, wherein the first and / or second detection agent is an antibody or an antigen-binding fragment thereof. 25. The method of any one of embodiments 1 to 23, wherein the first and / or second detection agent is an RNA-based binder molecule. 26. The method of any one of embodiments 1 to 25, wherein the sample comprises cells from a body fluid or tissue. 27. The method of embodiment 26, wherein the body fluid is blood, serum, or plasma. 28. The method of any one of embodiments 1 to 27, wherein the sample is derived from a patient. 29. The method of embodiment 28, wherein the patient is a mammal. 30. The method of embodiment 29, wherein the mammal is a human. 31. The method of any one of embodiments 1 to 30, wherein the sample comprises cells from an immortalized cell line. 32. A kit for carrying out the method according to any one of embodiments 1 to 31. 33. A sample of cells, prepared by the method according to any one of embodiments 1 to 31.

[0167] Specific embodiments of the present invention are described herein. Upon reading the foregoing description, it is expected that variations of the disclosed embodiments may become apparent to those skilled in the art, and such variations may be adopted as necessary. It is therefore intended that the present invention be practiced otherwise than as specifically described herein, and that the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto, as permitted by applicable law. Moreover, any combination of the above elements in all possible these variations is encompassed herein, unless otherwise noted herein or the context clearly contradicts. Many embodiments of the present invention have been described. However, it will be understood that various modifications may be made without departing from the spirit and scope of the present invention. Thus, the description in the Examples section is intended to illustrate, rather than limit, the scope of the invention as recited in the claims. EXAMPLES

[0168] The following is a description of various methods and materials used in the testing, which are set forth to provide those skilled in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure, nor are they intended to represent all of the experiments that have been performed and can be performed. It should be understood that the exemplary descriptions written in the present tense have not necessarily been performed, but rather the descriptions are those that can be performed to generate data and the like relevant to the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation should be taken into account.

[0169] Example 1: High-throughput screening method for antibodies suitable for tissue IHC The antibodies tested are shown in Table 1.

[0170] [Table 1]

[0171] NRK52E cells (Rattus norvegicus, kidney epithelial cells) were cultured and pelleted in collodion bags, then fixed in 10% NBF for 24 hours at room temperature. Collodion bags were generated by coating the entire inner surface of a 15 mL glass conical tube with l solution (Macron Chemicals catalogue no. 4560-04). After processing, a small pellet of cells was embedded in a paraffin block, and the remaining cells were removed from the collodion bag and transferred to xylene. After 3×15 min washing in xylene (until all visible paraffin was solubilized), the cells were hydrated by a successive ethanol gradient and then pelleted. The cells were resuspended in antigen retrieval solution and heated at 95° C. for 30 min by microwave radiation. After cooling, they were washed in PBS, counted and distributed into 96-well plates (30,000 / well). Plates with filter inserts can also be used for this step and are commercially available (Millipore, Burlington, MA). For example, the filter insert may have a pore size of 0.45 um to prevent loss of cells during washing and centrifugation of the plate.

[0172] ImmPRESS HRP Anti-Rabbit IgG Polymer Detection Kit (Vector Labs, Cat. No. MP-7451) was used. Cells were incubated with antibody solution for 60 minutes at room temperature with agitation. After washing and detection with appropriate secondary antibody, 50 uL of TMB solution was added per well and incubated for 30 minutes at room temperature. The color reaction was stopped and the optical density of each well was measured at 450 nm. Figure 2 shows the antibody binding in the form of optical density measured at 450 nm. Each antibody binding was tested in duplicate with 30,000 cells per well. The condition named "control negative" was for cells incubated with antibody diluent only.

[0173] For direct comparison, IHC assays were performed using the same antibody solutions on 4 um sections of the cell pellet blocks (IHC experiments were performed on a DAKO automated stainer). The H-score for each stained pellet was calculated based on an area quantification algorithm that calculates the total surface intensity staining of the cell pellet (Halo software, Indica Labs). The H-score for each pellet was calculated as follows: H-score = (surface area %1+) x 1 + (surface area %2+) x 2 + (% surface area %3+) x 3. Figures 3A-3B show the results of immunohistochemistry ("IHC") assays performed using various antibody solutions on 4 um sections of the cell pellet blocks. Figure 3A shows the IHC cell pellet staining for each antibody. Figure 3B shows the H-score for each stained pellet. The H-score for each stained pellet was calculated based on an area quantification algorithm that calculates the total surface intensity staining of the cell pellet.

[0174] Figure 4 shows the correlation assessment between the ELISA-like method provided herein and IHC staining. The Pearson correlation coefficient r = 0.929 (GraphPad Prism) indicated sufficient comparability between the screening results obtained by the high-throughput ELISA-like method and conventional IHC performed on formalin-fixed paraffin-embedded ("FFPE") tissue samples.

[0175] Example 2: Human Cell Materials and Methods Human cells are obtained, cultured, pelleted in collodion bags, and then fixed in 10% NBF at room temperature for 24 hours. The collodion bags are generated by coating the entire inner surface of a 15 mL glass conical tube with collodion solution (Macron Chemicals Catalog No. 4560-04). After processing, a small pellet of cells is embedded in a paraffin block, and the remaining cells are removed from the collodion bag and transferred to xylene. After washing in xylene for 3 x 15 minutes (until all visible paraffin is solubilized), the cells are hydrated by a continuous ethanol gradient and then pelleted. The cells are resuspended in antigen retrieval solution and heated at 95°C for 30 minutes by microwave radiation. After cooling, they are washed in PBS, counted, and distributed into 96-well plates (30,000 / well), some plates without inserts, and in some examples, plates with filter inserts are used, in which case the plates have a pore size of 0.45 um. Detection and quantification are performed as in Example 1.

[0176] Example 3: Non-human cell materials and methods Non-human cells are obtained, cultured, pelleted in collodion bags, and then fixed in 10% NBF at room temperature for 24 hours. Collodion bags are generated by coating the entire inner surface of a 15 mL glass conical tube with collodion solution (Macron Chemicals Catalog No. 4560-04). After processing, a small pellet of cells is embedded in a paraffin block, and the remaining cells are removed from the collodion bag and transferred to xylene. After washing in xylene for 3 x 15 minutes (until all visible paraffin is solubilized), the cells are hydrated through a continuous ethanol gradient and then pelleted. The cells are resuspended in antigen retrieval solution and heated in a microwave at 95°C for 30 minutes. After cooling, they are washed in PBS, counted, and distributed into 96-well plates (30,000 / well), some plates without inserts, and in some examples, plates with filter inserts are used, in which case the plates have a pore size of 0.45 um. Detection and quantification are performed as in Example 1.

[0177] Example 4: Human Tissue Cell Materials and Methods Human tissue containing cells is obtained, and cells from the tissue are cultured and pelleted in collodion bags, then fixed in 10% NBF at room temperature for 24 hours. Collodion bags are generated by coating the entire inner surface of a 15 mL glass conical tube with collodion solution (Macron Chemicals Catalog No. 4560-04). After processing, a small pellet of cells is embedded in a paraffin block, and the remaining cells are removed from the collodion bag and transferred to xylene. After washing in xylene for 3 x 15 minutes (until all visible paraffin is solubilized), the cells are hydrated by a continuous ethanol gradient and then pelleted. The cells are resuspended in antigen retrieval solution and heated at 95°C for 30 minutes by microwave radiation. After cooling, they are washed in PBS, counted, and distributed into 96-well plates (30,000 / well), some plates without inserts, and in some examples, plates with filter inserts are used, in which case the plates have a pore size of 0.45 um. Detection and quantification are performed as in Example 1.

[0178] Example 5: Non-human tissue cells materials and methods Non-human tissue containing cells is obtained, and cells from the tissue are cultured and pelleted in collodion bags, then fixed in 10% NBF at room temperature for 24 hours. Collodion bags are generated by coating the entire inner surface of a 15mL glass conical tube with collodion solution (Macron Chemicals catalog number 4560-04). After processing, a small pellet of cells is embedded in a paraffin block, and the remaining cells are removed from the collodion bag and transferred to xylene. After washing in xylene for 3x15 minutes (until all visible paraffin is solubilized), the cells are hydrated by a continuous ethanol gradient and then pelleted. The cells are resuspended in antigen retrieval solution and heated at 95°C for 30 minutes by microwave radiation. After cooling, they are washed in PBS, counted, and distributed into 96-well plates (30,000 / well), some plates without inserts, and in some instances plates with filter inserts are used, in which case the plates have a pore size of 0.45um. Detection and quantification are carried out as in Example 1.

[0179] Example 6: Detection of polynucleotides in cells The cells are obtained, cultured, pelleted in collodion bags, and then fixed in 10% NBF at room temperature for 24 hours. The collodion bags are generated by coating the entire inner surface of a 15 mL glass conical tube with collodion solution (Macron Chemicals Catalog No. 4560-04). After processing, a small pellet of cells is embedded in a paraffin block, and the remaining cells are removed from the collodion bag and transferred to xylene. After washing in xylene for 3 x 15 minutes (until all visible paraffin is solubilized), the cells are hydrated by a continuous ethanol gradient and then pelleted. The cells are resuspended in antigen retrieval solution and heated at 95°C for 30 minutes by microwave radiation. After cooling, they are washed in PBS, counted, and distributed into 96-well plates (30,000 / well), some plates without inserts, and in some examples, plates with filter inserts are used, in which case the plates have a pore size of 0.45 um. Detection and quantification are performed as in Example 1 to detect polynucleotides.

[0180] Example 7: Detection of non-polynucleotides in cells Cells are obtained, cultured, pelleted in collodion bags, and then fixed in 10% NBF at room temperature for 24 hours. Collodion bags are generated by coating the entire inner surface of a 15 mL glass conical tube with collodion solution (Macron Chemicals Catalog No. 4560-04). After processing, a small pellet of cells is embedded in a paraffin block, and the remaining cells are removed from the collodion bag and transferred to xylene. After washing in xylene for 3 x 15 minutes (until all visible paraffin is solubilized), the cells are hydrated by a continuous ethanol gradient and then pelleted. The cells are resuspended in antigen retrieval solution and heated at 95°C for 30 minutes by microwave radiation. After cooling, they are washed in PBS, counted, and distributed into 96-well plates (30,000 / well), some plates without inserts, and in some examples, plates with filter inserts are used, in which case the plates have a pore size of 0.45 um. Detection and quantification are performed as in Example 1 to detect molecules that are not polynucleotides.

[0181] Example 8: Immortalized Cell Materials and Methods Immortalized cells are obtained, cultured, pelleted in collodion bags, and then fixed in 10% NBF at room temperature for 24 hours. Collodion bags are generated by coating the entire inner surface of a 15 mL glass conical tube with collodion solution (Macron Chemicals Catalog No. 4560-04). After processing, a small pellet of cells is embedded in a paraffin block, and the remaining cells are removed from the collodion bag and transferred to xylene. After washing in xylene for 3 x 15 minutes (until all visible paraffin is solubilized), the cells are hydrated by a continuous ethanol gradient and then pelleted. The cells are resuspended in antigen retrieval solution and heated at 95°C for 30 minutes by microwave radiation. After cooling, they are washed in PBS, counted, and distributed into 96-well plates (30,000 / well), some plates without inserts, and in some examples, plates with filter inserts are used, in which case the plates have a pore size of 0.45 um. Detection and quantification are performed as in Example 1. * * * * *

[0182] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood therefore that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined herein.

Claims

**Claim 1** A method for detecting a molecule in a sample, comprising: a. obtaining cells from the sample; b. treating the cells with a fixative; c. paraffin-embedding the fixed cells; d. deparaffinizing and suspending the cells to obtain a single-cell suspension; e. contacting the suspended cells with a first detector that binds to at least one molecule of the suspended cells; contacting the cells bound to the first detector with a second detector; f. detecting the presence of the second detector bound to the cells of the sample, wherein detection of an amount of the second detector that exceeds the background bound to the sample indicates the presence of at least one molecule in the sample. **Claim 2** The method according to claim 1, wherein the molecule is a nucleic acid or a protein. **Claim 3** The method according to claim 2, wherein the nucleic acid is RNA. **Claim 4** The method according to claim 2, wherein the nucleic acid is DNA. **Claim 5** The method according to claim 1, further comprising removing unbound cells after contacting the suspended cells with the first detector. **Claim 6** The method according to claim 1, further comprising removing unbound second detector after contacting the cells bound to the first detector with the second detector. **Claim 7** The method according to claim 1, wherein the fixative is selected from the group consisting of formaldehyde, paraformaldehyde, glutaraldehyde, or neutral buffered formalin. **Claim 8** The method according to claim 7, wherein the fixative is neutral buffered formalin. **Claim 9** The method according to claim 8, wherein the neutral buffered formalin is 10% neutral buffered formalin. **Claim 10** The step of treating the cells with the fixative lasts for about 30 minutes to 60 minutes, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 7 hours, 7 hours to 8 hours, 8 hours to 9 hours, 9 hours to 10 hours, 10 hours to 11 hours, 11 hours to 12 hours, 12 hours to 13 hours, 13 hours to 14 hours, 14 hours to 15 hours, 15 hours to 16 hours, 16 hours to 17 hours, 17 hours to 18 hours, 18 hours to 19 hours, 19 hours to 20 hours, 20 hours to 21 hours, 21 hours to 22 hours, 22 hours to 23 hours, 23 hours to 24 hours, 24 hours to 36 hours, or 36 hours to 48 hours, according to the method of claim 1. **Claim 11** ​ The method according to claim 1, wherein the step of treating the cells with the fixative is performed at 4°C, room temperature, 40°C, or 60°C.

12. The method according to claim 1, wherein the step of treating the cells with the fixative is performed at room temperature for 24 hours.

13. The paraffin embedding of the fixed cells is a. contacting the cells with ethanol; b. contacting the cells with xylene; c. incubating the cells with paraffin, and the method according to claim 1.

14. The step of contacting the cells with ethanol is a. contacting the cells with 70% ethanol in water for 30 minutes; b. contacting the cells with 80% ethanol in water for 30 minutes; c. contacting the cells with 95% ethanol in water for 30 minutes; d. contacting the cells with 100% ethanol for 30 minutes, and the method according to claim 13.

15. The step of contacting the cells with xylene includes three exchanges of xylene for 20 minutes each, and the method according to claim 13.

16. The step of incubating the cells with paraffin includes four exchanges of paraffin for 20 minutes each, and the method according to claim 13.

17. The step of incubating the cells with paraffin is performed at 60°C, and the method according to claim 16.

18. The method according to claim 1, wherein the cells are deparaffinized by contacting the cells with xylene.

19. The cells are contacted with xylene for about 5 minutes to 10 minutes, 10 minutes to 15 minutes, 15 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 60 minutes, 60 minutes to 90 minutes, or 90 minutes to 120 minutes, and the method according to claim 18.

20. The cells are a. contacted with 70% ethanol in water for about 15 to about 30 minutes; b. contacted with 95% ethanol in water for about 15 to about 30 minutes; d. finally, contacted with 100% ethanol for about 15 to about 30 minutes, and further contacted with a continuous ethanol gradient, and the method according to claim 18.

21. The method according to claim 1, wherein the cells are resuspended in an antigen activation solution.

22. The method according to claim 21, wherein the cells are further heated at 95°C for about 30 minutes.

23. The method according to claim 22, wherein the cells are heated by microwave radiation. **Claim 24** The method according to claim 1, wherein the first and / or second detection agent is an antibody or an antigen-binding fragment thereof. **Claim 25** The method according to claim 1, wherein the first and / or second detection agent is an RNA-based binder molecule. **Claim 26** The method according to claim 1, wherein the sample comprises cells derived from a body fluid or tissue. **Claim 27** The method according to claim 26, wherein the body fluid is blood, serum, or plasma. **Claim 28** The method according to claim 1, wherein the sample is from a patient. **Claim 29** The method according to claim 28, wherein the patient is a mammal. **Claim 30** The method according to claim 29, wherein the mammal is a human. **Claim 31** The method according to claim 1, wherein the sample comprises cells derived from an immortalized cell line. **Claim 32** A kit for performing the method according to any one of claims 1 to 31. **Claim 33** A sample of cells prepared by the method according to any one of claims 1 to 31.

Images