Simultaneous imaging of nucleic acids and proteins in sample

Abstract

The present disclosure relates to simultaneous imaging of nucleic acids and proteins in a sample. In particular, the present disclosure provides compositions, methods, systems, and kits for imaging at least one target protein and at least one target nucleic acid in a single sample.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 421,390, filed on November 1, 2022, the entire content of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the simultaneous imaging of nucleic acids and proteins in a sample. Background Art

[0004] Analysis of nucleic acid and protein abundance and distribution is used to understand complex biological systems. Conventional strategies for detecting and characterizing nucleic acids and proteins, such as in situ hybridization, western blotting, and immunofluorescence detection, have contributed to the identification of target nucleic acids and proteins involved in the growth and development of whole organisms, as well as to specifically interrogate the causes and progression of various diseases. However, these strategies do not allow for the simultaneous detection of nucleic acids and proteins in a single sample. For example, the fixation and permeabilization techniques typically required for nucleic acid detection can prevent accurate protein detection in that same sample. Integrating multimodal analysis not only has the ability to enhance the discovery and characterization of potential correlations between nucleic acid (e.g., mRNA) abundance and distribution and target protein abundance and distribution, but can also facilitate the identification of cell phenotypes. Given the advantages associated with multimodal analysis, there is a need in the art for additional methods that allow for the simultaneous imaging of nucleic acids and proteins. Summary of the Invention

[0005] The present disclosure provides methods for imaging target proteins and target nucleic acids in a sample. In certain embodiments, the method comprises: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to a target protein in the sample, wherein the protein-binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (d) amplifying the target nucleic acid in the sample by performing an amplification process to produce amplicons; (e) (i) contacting the sample with a first labeled detection probe that comprises a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent, or (ii) contacting the sample with a first bridging oligonucleotide that comprises a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent, and contacting the sample with a first labeled detection probe that comprises a sequence complementary to the sequence of the first bridging oligonucleotide; (f) imaging the first labeled detection probe to detect the target protein; (g) (i) contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the amplicon, or (ii) contacting the sample with a second bridging oligonucleotide that comprises a sequence complementary to the sequence of the amplicon, and contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the second bridging oligonucleotide; and (h) imaging the second labeled detection probe to detect the target nucleic acid.

[0006] The present disclosure further provides a method for imaging target proteins and target nucleic acids in a sample, the method comprising: (a) binding a protein-binding reagent conjugated to an oligonucleotide to a target protein in the sample; (b) hybridizing a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) to the oligonucleotide conjugated to the protein-binding reagent; (c) amplifying the target nucleic acid in the sample by performing an amplification process to produce amplicons; (d) imaging the target protein by detecting a first labeled detection probe that hybridizes to the oligonucleotide conjugated to the protein-binding reagent or to a first bridging oligonucleotide that hybridizes to the oligonucleotide conjugated to the protein-binding reagent; and (e) imaging the target protein by detecting a second labeled detection probe that hybridizes to the amplicon or to a second bridging oligonucleotide that hybridizes to the amplicon.

[0007] In certain embodiments, the amplification process is a rolling circle amplification process. In certain embodiments, the rolling circle amplification process comprises: (a) contacting a sample with (i) a padlock probe comprising two nucleotide sequences complementary to a target nucleic acid and (ii) a ligase to generate a circular DNA template; and (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

[0008] In certain embodiments, the rolling circle amplification is initiated using a blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) complementary to an oligonucleotide conjugated to a protein binding reagent.

[0009] In certain embodiments, the protein binding reagent is an antibody or an antigen-binding fragment thereof.

[0010] In certain embodiments, the target nucleic acid comprises RNA.

[0011] In certain embodiments, the target nucleic acid is (i) an oligonucleotide conjugated to a protein binding reagent and / or (ii) a blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) complementary to an oligonucleotide conjugated to a protein binding reagent. In certain embodiments, the target nucleic acid is an oligonucleotide conjugated to a protein binding reagent. In certain embodiments, the target nucleic acid is a blocking oligonucleotide complementary to an oligonucleotide conjugated to a protein binding reagent, e.g., an exonuclease blocking oligonucleotide.

[0012] In certain embodiments, the nucleotide sequence complementary to the oligonucleotide conjugated to the protein binding reagent is located at the 5' end of the blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide). In certain embodiments, the oligonucleotide conjugated to the protein binding reagent comprises a barcode sequence, and wherein the blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) does not bind to the barcode sequence. In certain embodiments, the blocking oligonucleotide comprises one or more modified nucleotides. In certain embodiments, one or more modified nucleotides are located at the 3' end of the blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide). In certain embodiments, one or more modified nucleotides comprise nucleotides having phosphorothioate bonds.

[0013] In certain embodiments, providing the sample comprises (a) treating the sample with a fixative, (b) dehydrating the sample and / or (c) permeabilizing the sample. In certain embodiments, the sample is post-fixed prior to amplifying the target nucleic acid. In certain embodiments, the sample is permeabilized with hydrochloric acid prior to amplifying the target nucleic acid. In certain embodiments, the sample is treated with NHS-acetate prior to amplifying the target nucleic acid.

[0014] In certain embodiments, at least 10 target proteins are imaged in the sample. In certain embodiments, at least 10 target nucleic acids are imaged in the sample.

[0015] In certain embodiments, the sample is a tissue sample.

[0016] The present disclosure further provides kits for performing the methods disclosed herein. In certain embodiments, the kit includes at least one container containing blocking oligonucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagrams of exemplary methods according to the present disclosure are provided.

[0018] Figure 2 Schematic diagrams of exemplary methods according to the present disclosure are provided, wherein the method includes cyclic imaging.

[0019] Figure 3 Images of a sample stained for both protein and mRNA using an exemplary method according to the present disclosure are provided. The sample is stained for the CD97B protein and Cd79a RNA to label B cells, and for F4 / 80 to label macrophages.

[0020] Figure 4 Images are provided showing degradation of the 3' end of oligonucleotides conjugated to antibodies by Phi29 and protection of the 3' end of oligonucleotides using blocking oligonucleotides (e.g., exonuclease blocking oligonucleotides).

[0021] Figure 5 Images of a sample stained for both protein and mRNA over multiple imaging cycles using an exemplary method according to the present disclosure are provided. The sample is stained and imaged for the CD22 protein and mRNA, then for the CD4 protein and mRNA, and finally for the F4 / 80 protein and mRNA. DETAILED DESCRIPTION

[0022] The present disclosure relates to compositions and methods for simultaneous imaging of nucleic acids and proteins in a single sample. For example, the methods of the present disclosure include staining one or more proteins in a single sample, then staining one or more nucleic acids (e.g., mRNA), and then imaging both the proteins and the nucleic acids.

[0023] For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0024] I. Definitions;

[0025] II. Compositions and Methods for Simultaneous Imaging of Nucleic Acids and Proteins;

[0026] III. Systems and Kits; and

[0027] IV. Exemplary Embodiments.

[0028] I. Definitions

[0029] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. The following references provide one of ordinary skill in the art with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed., 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings ascribed to them below.

[0030] As used herein, when used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" or "an" can mean "one", but is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0031] The term "about" or "substantially" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with the practice in the art, "about" can mean three or more standard deviations. Alternatively, "about" can represent a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude of a particular value, preferably within 5-fold, more preferably within 2-fold.

[0032] The term "amplification process" generally refers to any process in which a portion of a nucleic acid is replicated or repeated into at least one additional nucleic acid molecule.

[0033] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0034] "Antibody fragment" refers to a molecule that comprises a portion of a full-length antibody and binds to the antigen to which the full-length antibody binds, other than the full-length antibody. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0035] As used herein, the terms "comprising," "including," "having," "containing," "may," "with," and variations thereof are intended to be open transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments "comprising" the embodiments or elements presented herein, "consisting of" and "consisting essentially of," whether explicitly recited or not.

[0036] The term "coupled" can refer to joining or associating two or more components by interaction, bond, linkage, force, or tie such that the two or more components are held together. In certain embodiments, the term "coupled" encompasses direct or indirect binding, where, for example, a first component binds directly to a second component or one or more intermediate molecules are disposed between the first and second components. Exemplary bonds include covalent bonds, ionic bonds, van der Waals interactions, and other bonds recognizable to those of skill in the art.

[0037] As used herein, the terms "detect / detection" mean determining the presence and / or existence of a target (e.g., a protein target or a nucleic acid target) in a limited portion of space, including but not limited to a sample. As used herein, the terms "detect / detection" can include determining the chemical and / or biological properties of the target, including but not limited to the ability to interact and in particular to bind to other compounds, the ability to activate another compound, and additional properties recognizable to those of skill in the art upon reading the present disclosure. The detection can be quantitative and / or qualitative. The detection is "quantitative" when it refers to, involves, or participates in the measurement of the quantity or amount of a target or signal (also referred to as quantification), which includes but is not limited to any analysis intended to determine the amount or proportion of a target or signal. The detection is "qualitative" when it refers to, involves, or participates in the identification of the quality or type of a target or signal in terms of its relative abundance with respect to another target or signal and is not quantified.

[0038] As used herein, the term "hybridization" refers to the process in which two single-stranded polynucleotides non-covalently bind to form a stable double-stranded polynucleotide.

[0039] As used herein, the term "imaging" refers to microscopy. In certain embodiments, microscopy includes immunofluorescence microscopy.

[0040] As used herein, the term "individual" or "subject" refers to a vertebrate or invertebrate, such as a human or non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, apes, and monkeys. In certain embodiments, the individual or subject is a human.

[0041] As used herein, a "label" refers to an agent that permits direct or indirect detection. Labels include, but are not limited to, fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels. Non-limiting examples of labels include green fluorescent protein ("GFP"), mCherry, dtTomato, or other fluorescent proteins known in the art (e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909 (2005), incorporated herein by reference), 32 P, 14 C, 125 I, 3 H and 131 I, fluorogens (such as rare earth chelates or fluorescein and its derivatives), Rhodamine and its derivatives, dansyl, umbelliferone, luciferases (such as firefly luciferase and bacterial luciferase) (U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydrophthalazine dione, and enzymes that produce a detectable signal, such as horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD)), and heterocyclic oxidases (such as uricase and xanthine oxidase).

[0042] As used herein, the term "ligation" refers to the formation of a covalent bond or linkage between the ends of two or more nucleic acids.

[0043] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody comprising the population is identical and / or binds the same epitope, except for possible variant antibodies (e.g., antibodies that contain naturally occurring mutations or that arise during the production of a monoclonal antibody preparation; such variants are typically present in minor amounts). In contrast to polyclonal antibody preparations, which generally include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies used in accordance with the presently disclosed subject matter can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals that contain all or part of the human immunoglobulin locus; such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0044] The term "nucleic acid" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide comprises a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by its base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. The term nucleic acid encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (e.g., messenger RNA (mRNA)), synthetic forms of DNA or RNA, and hybrid polymers that include two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acids described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleobases that have a derivatized sugar or phosphate backbone linkage or chemically modified residues.

[0045] The term "plurality" refers to a number greater than one. In certain embodiments, the term "plurality of proteins" refers to a number of proteins greater than one. For example, but not by way of limitation, a plurality of proteins includes at least two proteins. In certain embodiments, the term "plurality of nucleic acids" refers to a number of nucleic acids greater than one. For example, but not by way of limitation, a plurality of nucleic acids includes at least two nucleic acids.

[0046] The term "reverse transcription process" refers to the process of generating a complementary DNA strand using an enzyme called reverse transcriptase.

[0047] As used herein, the term "sample" refers to any sample containing one or more individual cells. In certain embodiments, the "sample" refers to a sample of biological material obtained from a subject, e.g., a tissue biopsy or tissue sample. In certain embodiments, the sample can be obtained from tissue, such as a tissue sample. Non-limiting examples of tissue include eye, muscle, skin, tendon, vein, artery, blood, heart, spleen, lymph node, bone, bone marrow, lung, bronchus, trachea, intestine, small intestine, large intestine, colon, rectum, salivary gland, tongue, gallbladder, appendix, liver, pancreas, brain, stomach, skin, kidney, ureter, bladder, urethra, gonad, testis, ovary, uterus, fallopian tube, thymus, pituitary gland, thyroid gland, adrenal gland or parathyroid tissue. In certain embodiments, the sample is obtained from a subject. In certain embodiments, the subject can be a human, a non-human primate (e.g., ape or monkey), a farm animal, a mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, a cat, a sheep, a pig, a goat, a cow or a horse. In certain embodiments, the subject is a human. In certain embodiments, the sample can be obtained from preserved tissue (e.g., fixed tissue), from frozen tissue or from fresh tissue (e.g., a tissue sample). In certain embodiments, samples that can be analyzed using the methods of the present disclosure include at least two or more cells. For example, but not by way of limitation, the sample can include about 10 or more cells, about 100 or more cells, about 1,000 or more cells, about 5,000 or more cells, about 10,000 or more cells, about 20,000 or more cells, about 30,000 or more cells, about 40,000 or more cells, about 50,000 or more cells, about 100,000 or more cells, about 150,000 or more cells, about 200,000 or more cells, about 300,000 or more cells, about 400,000 or more cells or 500,000 or more cells.

[0048] As used herein, the term "simultaneously" is not limited to two actions that occur simultaneously in a timely manner, but includes two actions that occur on a single sample, e.g., staining a sample for the presence of one or more target proteins and subsequently staining the same sample for the presence of one or more target nucleic acids, resulting in "simultaneous" staining of the sample for both the target nucleic acid and the target protein.

[0049] As used herein, the term "specifically binds" refers to the preferential binding of a target molecule (e.g., a protein or nucleic acid) relative to other molecules in the sample (e.g., proteins or nucleic acids).

[0050] II. Compositions and Methods for Simultaneous Imaging of Nucleic Acids and Proteins

[0051] The present disclosure relates to compositions and methods for simultaneously imaging nucleic acids and proteins in a sample. The methods of the present disclosure can be used for a variety of purposes. For example, the present disclosure provides methods for determining the spatial distribution of one or more proteins and one or more nucleic acids in a single sample. The methods of the present disclosure also allow visualization of both target nucleic acids (e.g., target DNA or target RNA) and target proteins in the same sample. In certain embodiments, the sample used in the methods of the present disclosure can contain multiple target nucleic acids, multiple target proteins, or both multiple target nucleic acids and target proteins. For example, but not by way of limitation, the methods of the present disclosure allow visualization of at least two or more target proteins (e.g., at least three or more, at least four or more, at least five or more, at least six or more, at least seven or more, at least eight or more, at least nine or more, or at least ten or more target proteins) and at least two or more target nucleic acids (e.g., at least three or more, at least four or more, at least five or more, at least six or more, at least seven or more, at least eight or more, at least nine or more, or at least ten or more target nucleic acids) in a single sample.

[0052] In certain embodiments, the compositions and methods of the present disclosure can also be used to determine specific characteristics of cells present in a sample. In certain embodiments, one or more target proteins can be present in cells in the sample that are different from one or more target nucleic acids. In certain embodiments, each of the target proteins among the multiple target proteins can be present in different cells within the sample and / or each of the target nucleic acids among the multiple target nucleic acids can be present in different cells within the sample. For example, but not by way of limitation, the compositions and methods of the present disclosure can be used to identify different cell types present in a sample.

[0053] In certain embodiments, the compositions and methods of the present disclosure allow multimodal analysis over time to facilitate detection of changes in cell characteristics and / or the presence of different cell types in samples collected at separate time points. For example, but not by way of limitation, the present disclosure can include obtaining samples (e.g., samples of tissue) at different time points and performing the methods of the present disclosure to visualize one or more target nucleic acids (e.g., one or more target DNA or RNA) and one or more target proteins in each of the samples to identify changes in the cell characteristics of the tissue over time. In certain embodiments, the present disclosure can include obtaining samples (e.g., samples of tissue) at different time points and performing the methods of the present disclosure to visualize one or more target nucleic acids (e.g., one or more target DNA or RNA) and one or more target proteins in each of the samples to identify changes in the cell types present in the tissue over time.

[0054] In certain embodiments, the compositions and methods of the present disclosure can be used for diagnostic purposes. For example, but not by way of limitation, the compositions and methods of the present disclosure can be used to determine the presence and / or absence of protein variants and / or nucleic acid variants associated with a disease, such as for diagnosing a subject having the disease. In certain embodiments, the compositions and methods of the present disclosure can be used for pathological anatomical tissues, e.g., to identify a disease. Further, the compositions and methods of the present disclosure allow for multimodal analysis over time to facilitate diagnostic assessment.

[0055] Figure 1 A flowchart of an exemplary method of the present disclosure is provided. In certain embodiments, the method of the present disclosure can include providing a sample, detecting one or more target proteins in the sample, detecting one or more target nucleic acids in the sample, and imaging one or more target proteins and one or more target nucleic acids in the sample. In certain embodiments, providing a sample includes preparing the sample.

[0056] A. Sample Preparation

[0057] As Figure 1 shown, the method of the present disclosure includes preparing a sample for imaging one or more target proteins and one or more target nucleic acids in the sample.

[0058] In certain embodiments, the sample to be analyzed can be prepared prior to detecting one or more target proteins and one or more target nucleic acids. In certain embodiments, such sample preparation can include a fixation process, a permeabilization process, a dehydration process, a rehydration process, a post-fixation process, and / or a nuclease inhibition process. In certain embodiments, sample preparation includes a fixation process. In certain embodiments, sample preparation includes a fixation process and a dehydration process. In certain embodiments, sample preparation can further include a permeabilization process. In certain embodiments, sample preparation further includes a rehydration process. In certain embodiments, sample preparation includes a fixation process, a dehydration process, and a rehydration process. In certain embodiments, sample preparation includes a fixation process, a dehydration process, a permeabilization process, and a rehydration process. In certain embodiments, sample preparation further includes a post-fixation process. In certain embodiments, sample preparation includes a fixation process, a dehydration process, a permeabilization process, a rehydration process, and a post-fixation process. In certain embodiments, sample preparation further includes a nuclease inhibition process. In certain embodiments, sample preparation includes a fixation process, a dehydration process, a permeabilization process, a rehydration process, a post-fixation process, and a nuclease inhibition process.

[0059] In certain embodiments, the fixation process includes contacting the sample with a fixative. Non-limiting examples of fixatives include aldehydes (e.g., formaldehyde, paraformaldehyde, and glutaraldehyde), imidoesters, N-hydroxysuccinimide (NHS) esters (e.g., bis-NHS esters), alcohols (e.g., methanol and ethanol), acetone, and acetic acid. In certain embodiments, the fixative is formaldehyde. In certain embodiments, the fixative includes two or more fixatives. For example, the fixative can include formaldehyde and glutaraldehyde. In certain embodiments, the sample is fixed at a final fixative concentration of about 0.1% to about 10%, about 1% to about 10%, about 1% to about 8%, about 2% to about 7%, about 3% to about 6%, or about 3% to about 5%. In certain embodiments, the sample is fixed at a final fixative concentration of about 3% to about 6%. In certain embodiments, the sample is fixed at a final fixative concentration of about 3% to about 5%. In certain embodiments, the sample is fixed at a final fixative concentration of about 4%. In certain embodiments, the sample is fixed at a final formaldehyde concentration of about 0.1% to about 10%, about 1% to about 10%, about 1% to about 8%, about 2% to about 7%, about 3% to about 6%, or about 3% to about 5%, such as about 4%. In certain embodiments, the sample can be contacted with the fixative for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, or about 5 minutes or less. In certain embodiments, the sample can be contacted with the fixative for about 5 minutes to about 1 hour, such as about 5 minutes to about 30 minutes. In certain embodiments, the sample can be contacted with the fixative for about 5 minutes to about 1 hour. In certain embodiments, the sample can be contacted with the fixative for about 5 minutes to about 30 minutes. In certain embodiments, the sample can be contacted with the fixative at a temperature in the range of about 0°C to 50°C, such as at room temperature (RT). In certain embodiments, the method can include a post-fixative process. For example, the sample can be fixed after contacting the sample with the protein-binding reagent but before detecting the target nucleic acid in the sample. The fixatives disclosed herein can be used for post-fixing the sample. In certain embodiments, the sample can be post-fixed at a final fixative concentration of about 3% to about 6%, for example, at a final fixative concentration of about 4%. In certain embodiments, the post-fixative includes two or more fixatives, for example, formaldehyde (e.g., at a concentration of about 3% to about 6%, such as about 4%) and glutaraldehyde (e.g., at a concentration of about 0.5% to about 2%, such as about 1%).

[0060] In certain embodiments, sample preparation may include a dehydration process. In certain embodiments, the dehydration process results in a reduction in the amount of water in the sample. In certain embodiments, such dehydration is achieved by contacting the sample with an alcohol (e.g., an alcohol series). In certain embodiments, dehydration includes contacting the sample with a solution having an increasing alcohol content. For example, but not by way of limitation, the dehydration process may include contacting the sample with an ethanol series, wherein the sample is contacted with ethanol solutions of increasing concentration. In certain embodiments, the sample may be contacted with each concentration of alcohol (e.g., ethanol) in an alcohol series (e.g., an ethanol series) for about 0.5 minutes to about 1 hour, e.g., about 1 minute. In certain embodiments, the ethanol series includes 70%, 75%, 80%, 85%, 90%, 95% and / or 100% ethanol, e.g., 70%, 85% and / or 100% ethanol. In certain embodiments, the ethanol series includes 70%, 85% and 100% ethanol. In certain embodiments, the dehydration process includes contacting the sample with 70% ethanol, subsequently contacting the sample with 85% ethanol, and then contacting the sample with 100% ethanol. In certain embodiments, the sample may be contacted with the alcohol (e.g., an alcohol in an alcohol series) at a temperature in the range of about 0°C to 50°C, e.g., at room temperature (RT). In certain embodiments, the sample may be contacted with each different concentration of alcohol (e.g., ethanol) for about 0.5 minutes to about 1 hour, e.g., about 0.5 minutes to about 10 minutes, e.g., about 1 minute.

[0061] In certain embodiments, the sample can be permeabilized prior to contacting the sample with a protein binding reagent for detecting a target protein. For example, but not by way of limitation, the sample can be permeabilized after sample fixation and prior to contacting the sample with the protein binding reagent. Techniques for permeabilizing cells are known in the art, and one of ordinary skill in the art will be able to evaluate the appropriateness of a particular technique for use in conjunction with the methods of the present disclosure. Non-limiting examples of reagents for permeabilizing cells include detergents (e.g., saponin, Tween-20, and Triton X-100) and fixatives (e.g., acetone, methanol, and ethanol). For example, but not by way of limitation, the sample can be permeabilized with an alcohol (e.g., methanol) and / or a detergent (e.g., such as Triton X-100). In certain embodiments, the sample is permeabilized with a detergent. In certain embodiments, the sample is permeabilized with a fixative. In certain embodiments, the reagent for permeabilization can be used at a concentration of about 0.1% to about 10%, e.g., about 0.1% to about 10%, about 0.1% to about 10%, about 0.1% to about 10%, about 0.1% to about 10%, about 0.1% to about 10%, about 0.1% to about 10%. In certain embodiments, the reagent for permeabilization (e.g., a detergent) can be used at a concentration of about 0.1% to about 1.0%, e.g., 0.5%. In certain embodiments, permeabilization can be carried out by contacting the fixed sample with 0.5% Triton X-100. In certain embodiments, the sample can be contacted with the permeabilization reagent for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, about 30 minutes or less, about 20 minutes or less, about 10 minutes or less, or about 5 minutes or less. In certain embodiments, the sample can be contacted with the permeabilization reagent, e.g., Triton X-100, for about 20 minutes. In certain embodiments, the sample can be contacted with the permeabilization reagent (e.g., Triton X-100) at a temperature in the range of about 0°C to 50°C, e.g., at room temperature (RT).

[0062] B. Protein Staining

[0063] As Figure 1As shown, the method of the present disclosure may include staining and imaging one or more target proteins in a sample. For example, but not by way of limitation, the method of the present disclosure may include staining and imaging one target protein, two or more target proteins, three or more target proteins, four or more target proteins, five or more target proteins, six or more target proteins, seven or more target proteins, eight or more target proteins, nine or more target proteins, or ten or more target proteins. In certain embodiments, at least 5 target proteins, at least 10 target proteins, at least 15 target proteins, at least 20 target proteins, at least 35 target proteins, at least 40 target proteins, at least 45 target proteins, at least 50 target proteins, at least 55 target proteins, at least 60 target proteins, at least 65 target proteins, at least 70 target proteins, at least 75 target proteins, at least 80 target proteins, at least 85 target proteins, at least 90 target proteins, at least 95 target proteins, at least 100 target proteins, at least 105 target proteins, at least 110 target proteins, at least 115 target proteins, at least 120 target proteins, at least 125 target proteins, at least 130 target proteins, at least 135 target proteins, at least 140 target proteins, at least 145 target proteins, at least 150 target proteins, at least 155 target proteins, at least 160 target proteins, at least 165 target proteins, at least 170 target proteins, at least 175 target proteins, at least 180 target proteins, at least 185 target proteins, at least 190 target proteins, at least 195 target proteins, or at least 200 target proteins are imaged in a single sample using the presently disclosed method. In certain embodiments, about 15 to about 100 target proteins are stained and imaged in a single sample using the presently disclosed method. In certain embodiments, about 15 to about 30 target proteins are stained and imaged in a single sample using the presently disclosed method. In certain embodiments, one or more target proteins may be present in the same cells within the sample. Alternatively, one or more target proteins may be present within different cells (e.g., different cell types) within the sample (e.g., as shown in Figure 3 , Figure 4 and Figure 5 ).

[0064] In certain embodiments, proteins that can be stained and imaged using the methods of the present disclosure include any protein present on or at the cell surface. For example, but not by way of limitation, the target protein can be an intracellular protein, an extracellular protein, or a transmembrane protein. In certain embodiments, the target protein is a mutant form of the protein or the wild-type form of the protein. In certain embodiments, the target protein is an exogenous protein, e.g., a protein exogenously expressed in a sample. In certain embodiments, the target protein is an endogenous protein, e.g., a protein endogenously expressed in a sample. In certain embodiments, the target protein is a post-translationally modified form of the protein.

[0065] In certain embodiments, staining and imaging of one or more protein targets in a sample can include contacting the sample with a reagent that binds to the target protein in the sample (also referred to herein as a "protein-binding reagent"). In certain embodiments, the protein-binding reagent is a reagent that specifically binds to the target protein, e.g., specifically binds to the target protein of cells in the sample. In certain embodiments, the reagent that binds to the target protein permits imaging of the target protein. In certain embodiments, the reagent that binds to the target protein permits quantitative analysis of the target protein. Non-limiting examples of protein-binding reagents include antibodies (or antigen-binding fragments thereof), aptamers, affimers, peptides, and small molecules.

[0066] In certain embodiments, the protein-binding reagent is an antibody (or antigen-binding fragment thereof) specific for the target protein. In certain embodiments, the affinity between the antibody (or antigen-binding fragment thereof) and the target protein is characterized by a dissociation constant (K d ) of ≤1 M, ≤100 mM, ≤10 mM, ≤1 mM, ≤100 μM, ≤10 μM, ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM. In certain embodiments, an antibody specific for the target protein can have a K -3 of about 10 -8 or less or 10 -8 M or less (e.g., 10 -13 M to 10 -9 M, e.g., 10 -13 M to 10 d . In certain embodiments, the antibody can be an antibody fragment as described herein. For example, but not by way of limitation, the antibody can be Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, diabody, or single-domain antibody. In certain embodiments, the antibody can be a humanized or chimeric antibody.

[0067] In certain embodiments, a protein binding reagent is conjugated to an oligonucleotide. In certain embodiments, an antibody is conjugated to an oligonucleotide, which is also referred to herein as an “antibody-oligonucleotide conjugate”. Exemplary antibody-oligonucleotide conjugates are shown and used in Figure 1 , Figure 2 , Figure 4 and Figure 5 . In certain embodiments, the oligonucleotide conjugated to a protein binding reagent (e.g., an antibody or an antigen-binding fragment thereof) can be from about 5 to about 200 nucleotides in length, e.g., from about 5 to about 150 nucleotides in length, from about 5 to about 100 nucleotides in length, from about 5 to about 50 nucleotides in length, from about 10 to about 150 nucleotides in length, from about 20 to about 100 nucleotides in length, or from about 10 to about 100 nucleotides in length. In certain embodiments, the oligonucleotide conjugated to an antibody can be from about 5 to about 50 nucleotides in length, e.g., from about 5 to about 45 nucleotides in length, from about 5 to about 40 nucleotides in length, from about 5 to about 35 nucleotides in length, from about 5 to about 30 nucleotides in length, from about 5 to about 25 nucleotides in length, from about 5 to about 20 nucleotides in length, from about 5 to about 15 nucleotides in length, from about 5 to about 10 nucleotides in length, from about 10 to about 50 nucleotides in length, from about 15 to about 50 nucleotides in length, from about 20 to about 50 nucleotides in length, from about 25 to about 50 nucleotides in length, from about 30 to about 50 nucleotides in length, from about 35 to about 50 nucleotides in length, from about 40 to about 50 nucleotides in length, from about 10 to about 40 nucleotides in length, or from about 10 to about 30 nucleotides in length. In certain embodiments, the antibody can be a TotalSeq TM antibody (BioLegend, San Diego, CA). In certain embodiments, the use of an antibody-oligonucleotide conjugate allows for imaging of a protein by binding a detection probe to the oligonucleotide conjugated to the antibody and / or a bridging oligonucleotide that binds to the oligonucleotide conjugated to the antibody. In certain embodiments, the use of an antibody-oligonucleotide conjugate allows for imaging of a protein by amplifying the oligonucleotide conjugated to the antibody and subsequently detecting the resulting amplicon by binding a detection probe to the amplicon and / or by detecting a bridging oligonucleotide that binds to the resulting amplicon. Alternatively, the use of an antibody-oligonucleotide conjugate allows for imaging of a protein by amplifying a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that binds to the oligonucleotide conjugated to the antibody and subsequently detecting the resulting amplicon by binding a detection probe to the amplicon and / or by detecting a bridging oligonucleotide that binds to the resulting amplicon.

[0068] In certain embodiments, the oligonucleotide conjugated to a protein binding reagent can include, for example, asFigure 1 The barcodes shown. In certain embodiments, the barcode is a unique nucleotide sequence that can be used to identify an antibody conjugated to the barcode. In certain embodiments, the barcode is from about 10 to about 50 nucleotides in length, such as from about 10 to about 30 nucleotides. In certain embodiments, the barcode is from about 10 to about 20 nucleotides in length. In certain embodiments, the barcode is about 15 nucleotides in length. In certain embodiments, a detection probe can bind to the barcode. For example, the detection probe can comprise a sequence that is at least partially complementary to the barcode. Alternatively or additionally, the detection probe can bind to a bridging oligonucleotide that binds to the barcode. For example, the detection probe can comprise a sequence that is at least partially complementary to the bridging oligonucleotide that is at least partially complementary to the barcode, as Figure 1 shown.

[0069] In certain embodiments, the oligonucleotide conjugated to the protein binding reagent can further comprise a primer sequence, such as, as Figure 1 shown. In certain embodiments, the primer sequence can be used to amplify the oligonucleotide. For example, but not by way of limitation, the primer sequence present in the oligonucleotide conjugated to the protein binding reagent can be used to amplify the oligonucleotide during an amplification reaction for amplifying a target nucleic acid in a sample. In certain embodiments, the primer sequence is from about 10 to about 50 nucleotides in length, such as from about 10 to about 30 nucleotides. In certain embodiments, the barcode is about 20 nucleotides in length.

[0070] In certain embodiments, multiple protein binding reagents for binding multiple different target proteins can be used in the present disclosure. For example, but not by way of limitation, each protein binding reagent, such as each antibody-oligonucleotide conjugate, specifically binds to a single target protein. In certain embodiments, two or more protein binding reagents, three or more protein binding reagents, four or more protein binding reagents, five or more protein binding reagents, six or more protein binding reagents, seven or more protein binding reagents, eight or more protein binding reagents, nine or more protein binding reagents, or ten or more protein binding reagents can be used in the present disclosure, wherein each protein binding reagent specifically binds to a single target protein. In certain embodiments, at least 5 protein binding reagents, at least 10 protein binding reagents, at least 15 protein binding reagents, at least 20 protein binding reagents, at least 35 protein binding reagents, at least 40 protein binding reagents, at least 45 protein binding reagents, at least 50 protein binding reagents, at least 55 protein binding reagents, at least 60 protein binding reagents, at least 65 protein binding reagents, at least 70 protein binding reagents, at least 75 protein binding reagents, at least 80 protein binding reagents, at least 85 protein binding reagents, at least 90 protein binding reagents, at least 95 protein binding reagents, at least 100 protein binding reagents, at least 105 protein binding reagents, at least 110 protein binding reagents, at least 115 protein binding reagents, at least 120 protein binding reagents, at least 125 protein binding reagents, at least 130 protein binding reagents, at least 135 protein binding reagents, at least 140 protein binding reagents, at least 145 protein binding reagents, at least 150 protein binding reagents, at least 155 protein binding reagents, at least 160 protein binding reagents, at least 165 protein binding reagents, at least 170 protein binding reagents, at least 175 protein binding reagents, at least 180 protein binding reagents, at least 185 protein binding reagents, at least 190 protein binding reagents, at least 195 protein binding reagents, or at least 200 protein binding reagents can be used to detect target proteins in a single sample by the methods of the present disclosure. In certain embodiments, about 15 to about 100 protein binding reagents can be used to detect target proteins in a single sample by the methods of the present disclosure. In certain embodiments, about 15 to about 30 protein binding reagents can be used to detect target proteins in a single sample by the methods of the present disclosure. The use of multiple protein binding reagents, such as antibody-oligonucleotide conjugates, allows for imaging of multiple proteins in a single sample and can also allow for analysis of the spatial position of each protein relative to one another.

[0071] In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) for a period of time and under conditions that support specific binding of the protein binding reagent to the target protein. In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) for about 24 hours or less, about 23 hours or less, about 22 hours or less, about 21 hours or less, about 20 hours or less, about 19 hours or less, about 18 hours or less, about 17 hours or less, about 16 hours or less, about 15 hours or less, about 14 hours or less, about 13 hours or less, about 12 hours or less, about 11 hours or less, about 10 hours or less, about 9 hours or less, about 8 hours or less, about 7 hours or less, about 6 hours or less, about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less or about 60 minutes or less. In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) for about 1 to about 48 hours, such as about 1 to about 42 hours, about 1 to about 40 hours, about 1 to about 38 hours, about 1 to about 36 hours, about 1 to about 34 hours, about 1 to about 32 hours, about 1 to about 30 hours, about 1 to about 28 hours, about 1 to about 26 hours, about 1 to about 24 hours, about 1 to about 22 hours, about 1 to about 20 hours, about 1 to about 18 hours, about 1 to about 16 hours, about 1 to about 14 hours, about 1 to about 12 hours, about 1 to about 10 hours, about 1 to about 8 hours, about 1 to about 6 hours, about 1 to about 4 hours or about 1 to about 2 hours. In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) for about 1 to about 24 hours. In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) for about 1 to about 20 hours. In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) for about 1 to about 16 hours. In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) for about 1 to about 12 hours. In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) for about 1 to about 8 hours. In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) for about 1 to about 3 hours. In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) at a temperature in the range of about 0°C to 50°C, such as at room temperature (RT). In certain embodiments, the sample can be contacted with a protein binding reagent (e.g., an antibody specific for the target protein) at a temperature in the range of about 0°C to 10°C, such as at 4°C.

[0072] In certain embodiments, contacting the protein binding reagent with the sample can include performing a blocking step to reduce background noise. In certain embodiments, the blocking step includes contacting the sample with one or more oligonucleotides (e.g., blocking oligonucleotides) that are complementary to one or more nucleotide sequences (e.g., two or more, three or more, four or more, or five or more nucleotide sequences) present in the oligonucleotide conjugated to the antibody. In certain embodiments, the blocking oligonucleotide can be complementary to a conserved sequence present in the oligonucleotide conjugated to the protein binding reagent (e.g., a conserved sequence present in each oligonucleotide that binds to multiple protein binding reagents used in the disclosed methods). In certain embodiments, the blocking oligonucleotide has a length of from about 10 to about 100 nucleotides, such as from about 10 to about 50 nucleotides. In certain embodiments, the blocking step can be performed prior to incubating the protein binding reagent with the sample. Alternatively, the blocking step can be performed simultaneously with incubating the protein binding reagent with the sample. In certain embodiments, the blocking step can be performed after incubating the protein binding reagent with the sample. In certain embodiments, the blocking step can be performed for about 5 minutes to 24 hours, such as, about 15 minutes to about 24 hours, about 15 minutes to about 20 hours, about 15 minutes to about 16 hours, about 15 minutes to about 12 hours, about 15 minutes to about 8 hours, about 15 minutes to about 4 hours, about 15 minutes to about 1 hour, about 1 hour to about 24 hours, about 2 hours to about 24 hours, about 3 hours to about 24 hours, about 4 hours to about 24 hours, about 5 hours to about 24 hours, about 6 hours to about 24 hours, about 7 hours to about 24 hours, about 8 hours to about 24 hours, about 9 hours to about 24 hours, about 10 hours to about 24 hours, about 12 hours to about 24 hours, about 14 hours to about 24 hours, about 16 hours to about 24 hours, about 18 hours to about 24 hours, about 20 hours to about 24 hours, about 22 hours to about 24 hours, about 2 hours to about 12 hours, or about 2 hours to about 6 hours. In certain embodiments, the blocking step can be performed for about 1 to about 24 hours. In certain embodiments, the blocking step can be performed for about 1 to about 20 hours. In certain embodiments, the blocking step can be performed for about 1 to about 16 hours. In certain embodiments, the blocking step can be performed for about 1 to about 12 hours. In certain embodiments, the blocking step can be performed for about 1 to about 8 hours. In certain embodiments, the blocking step can be performed for about 1 to about 3 hours. In certain embodiments, the blocking step can be performed at a temperature in the range of about 0°C to 25°C, such as at 4°C or at room temperature.

[0073] In certain embodiments, the sample to be analyzed by the methods of the present disclosure can be crosslinked. For example, but not by way of limitation, the sample can be crosslinked prior to detecting nucleic acids in the sample. Crosslinking of the sample can be achieved by exposing the cells to any crosslinking agent. In certain embodiments, the fixatives as disclosed herein can be used for crosslinking. In certain embodiments, crosslinking can be performed by exposing the sample to formaldehyde. In certain embodiments, crosslinking can be performed by exposing the sample to glutaraldehyde. In certain embodiments, crosslinking can be performed by exposing the sample to a solution comprising formaldehyde and glutaraldehyde. In certain embodiments, crosslinking can be performed by exposing the sample to a solution comprising from about 1% to about 10%, such as about 4%, paraformaldehyde and from about 0.1% to about 5%, such as about 1%, glutaraldehyde. In certain embodiments, crosslinking is performed as a post-fixation step as described herein. In certain embodiments, crosslinking is performed to maintain the binding of the target protein to the protein binding reagent during the sample processing steps required to prepare the sample for nucleic acid detection. For example, but not by way of limitation, HCl used to permeabilize the cells for nucleic acid detection abolishes the binding of the target protein to the protein binding reagent, and crosslinking prior to HCl treatment prevents this from occurring, thus allowing for the simultaneous detection of proteins and nucleic acids in a single sample.

[0074] C. Nucleic Acid Staining

[0075] In certain embodiments, the methods of the present disclosure can further comprise staining and imaging one or more nucleic acids in the sample. For example, but not by way of limitation, the methods of the present disclosure can further comprise staining and / or imaging one or more nucleic acids in the sample before and / or after staining and / or imaging one or more proteins in the sample. In certain embodiments, the methods of the present disclosure can further comprise staining one or more nucleic acids in the sample before or after staining one or more proteins in the sample. In certain embodiments, the methods of the present disclosure can further comprise imaging one or more nucleic acids in the sample before or after imaging one or more proteins in the sample. In certain embodiments, the methods of the present disclosure can further comprise staining one or more nucleic acids in the sample after staining one or more proteins in the sample as described in Example 1. In certain embodiments, the methods of the present disclosure can further comprise imaging one or more nucleic acids in the sample after imaging one or more proteins in the sample as described in Example 1.

[0076] In certain embodiments, the methods of the present disclosure may further include staining and imaging one target nucleic acid, two or more target nucleic acids, three or more target nucleic acids, four or more target nucleic acids, five or more target nucleic acids, six or more target nucleic acids, seven or more target nucleic acids, eight or more target nucleic acids, nine or more target nucleic acids, or ten or more target nucleic acids. In certain embodiments, at least 5 target nucleic acids, at least 10 target nucleic acids, at least 15 target nucleic acids, at least 20 target nucleic acids, at least 35 target nucleic acids, at least 40 target nucleic acids, at least 45 target nucleic acids, at least 50 target nucleic acids, at least 55 target nucleic acids, at least 60 target nucleic acids, at least 65 target nucleic acids, at least 70 target nucleic acids, at least 75 target nucleic acids, at least 80 target nucleic acids, at least 85 target nucleic acids, at least 90 target nucleic acids, at least 95 target nucleic acids, or at least 100 target nucleic acids are stained and imaged in a single sample using the presently disclosed methods. In certain embodiments, at least about 100 target nucleic acids, at least about 200 target nucleic acids, at least about 300 target nucleic acids, at least about 400 target nucleic acids, at least about 500 target nucleic acids, at least about 600 target nucleic acids, at least about 700 target nucleic acids, at least about 800 target nucleic acids, at least about 900 target nucleic acids, at least about 1,000 target nucleic acids, at least about 1,500 target nucleic acids, at least about 2,000 target nucleic acids, at least about 2,500 target nucleic acids, at least about 3,000 target nucleic acids, at least about 3,500 target nucleic acids, at least about 4,000 target nucleic acids, at least about 4,500 target nucleic acids, or at least about 5,000 target nucleic acids are stained and imaged in a single sample using the presently disclosed methods. In certain embodiments, about 100 target nucleic acids are stained and imaged in a single sample using the presently disclosed methods.

[0077] In certain embodiments, the target nucleic acid can be any nucleic acid molecule present in the sample to be analyzed (e.g., a DNA molecule or an RNA molecule). In certain embodiments, the target nucleic acid is mRNA. In certain embodiments, the target nucleic acid is a non-coding RNA, such as tRNA, rRNA, or microRNA (miRNA). In certain embodiments, the target nucleic acid is a DNA molecule. In certain embodiments, the target nucleic acid is genomic DNA. In certain embodiments, the target nucleic acid is exogenous nucleic acid, such as viral nucleic acid. In certain embodiments, the target nucleic acid is a variant of the target nucleic acid. In certain embodiments, the target nucleic acid is engineered barcoded RNA. In certain embodiments, the target nucleic acid is a guide RNA, for example, that can be used in gene editing techniques. In certain embodiments, the target nucleic acid is a guide RNA used with a CRISPR enzyme.

[0078] In certain embodiments, the target nucleic acid is an oligonucleotide conjugated to a protein binding reagent and / or a blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) complementary to an oligonucleotide conjugated to a protein binding reagent. For example, but not by way of limitation, the target nucleic acid is an oligonucleotide conjugated to a protein binding reagent used in a method for detecting a target protein. For example, the target nucleic acid is the oligonucleotide of an antibody-oligonucleotide conjugate. In certain embodiments, the target nucleic acid is a blocking oligonucleotide complementary to an oligonucleotide conjugated to a protein binding reagent, such as an exonuclease blocking oligonucleotide. In certain embodiments, the target nucleic acid is a blocking oligonucleotide complementary to the oligonucleotide of an antibody-oligonucleotide conjugate, such as an exonuclease blocking oligonucleotide.

[0079] Target nucleic acids stained and imaged using the disclosed methods can have different lengths. In certain embodiments, the target nucleic acid is about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 60 or more, about 80 or more, about 100 or more, about 150 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 1,000 or more, about 5,000 or more, or about 10,000 or more nucleotides in length. In certain embodiments, the target nucleic acid comprises 10 or more contiguous nucleotides of a known sequence. For example, but not by way of limitation, the target nucleic acid can comprise about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 60 or more, about 80 or more, about 100 or more, about 150 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, about 1,000 or more, about 5,000 or more, or about 10,000 or more contiguous nucleotides of a known sequence. In certain embodiments, the target nucleic acid comprises 10 or more contiguous nucleotides of an unknown sequence.

[0080] In certain embodiments, staining and imaging of nucleic acids in a sample includes performing an amplification process to amplify a target nucleic acid. One of ordinary skill in the art can evaluate suitable nucleic acid amplification methods known in the art to identify strategies suitable for amplifying the target nucleic acid. Non-limiting examples of such amplification methods include polymerase chain reaction (PCR), reverse transcriptase PCR, real-time PCR, rolling circle amplification (RCA), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), transcription-mediated amplification (TMA), single primer isothermal amplification (SPIA), helicase-dependent amplification (HDA), loop-mediated amplification (LAMP), recombinase-polymerase amplification (RPA), nicking enzyme amplification reaction (NEAR), nick endonuclease-assisted nanoparticle activation (NENNA), and ligase chain reaction (LCR). Fakruddin et al., J. Pharm. Bioallied. Sci. 5(4):245-252 (2013) and Yan et al., Mol. BioSyst. 10:970-1003 (2014) disclose additional amplification methods for the present disclosure, the content of each of which is hereby incorporated by reference in its entirety. In certain embodiments, the amplification process is RCA.

[0081] In certain embodiments, the amplification process includes an amplification reaction using a polymerase having exonuclease activity, e.g., a polymerase having 3'→5' exonuclease activity. In certain embodiments, the amplification reaction includes an amplification reaction comprising a polymerase having strand displacement activity. Non-limiting examples of polymerases having strand displacement and / or exonuclease activity include Bst polymerase, DNA polymerase ε (Polε), DNA polymerase δ (Polδ), and Phi29 polymerase and derivatives thereof. In certain embodiments, derivatives of Phi29 polymerase include Phi29 polymerase comprising one or more modifications, e.g., amino acid mutations, compared to wild-type Phi29 polymerase. In certain embodiments, the Phi29 polymerase is EquiPhi29 TM (Thermo Scientific). In certain embodiments, the amplification process includes an amplification reaction comprising Phi29 polymerase.

[0082] In certain embodiments, performing the amplification process includes contacting the sample with reagents required for the amplification process and performing the process under conditions suitable for amplifying the target nucleic acid. Non-limiting examples of such reagents include polymerases, reverse transcriptases, nucleoside triphosphates or NTP analogs, primers, probes, primers, cofactors, ligation reaction reagents, endonucleases, lysis reagents, dyes, markers, or labels. In certain embodiments, additional reagents may include RNase inhibitors to protect the integrity of RNA in the sample, e.g., by inhibiting the activity of RNase A, B, and / or C.

[0083] In certain embodiments, nucleic acid staining and imaging in a sample can be performed using padlock probes, i.e., linear probes, which can be converted into circular DNA molecules by ligation after hybridization to a target nucleic acid (e.g., target mRNA). In certain embodiments, a ligase that ligates single-stranded DNA, e.g., SplintR ligase, is used for ligation. Additional disclosure regarding the use of padlock oligonucleotides is provided in Sountoulidis et al., PLoS Biology 18(11):e3000675 (2020), the content of which is incorporated herein by reference.

[0084] In certain embodiments, hybridization of the padlock oligonucleotide to the target nucleic acid can occur in a hybridization buffer for a period of about 1 hour to about 24 hours, e.g., a period of about 2 hours to about 20 hours, about 2 hours to about 16 hours, or about 2 hours to about 12 hours. In certain embodiments, hybridization of the padlock oligonucleotide to the target nucleic acid can occur in a hybridization buffer for a period of about 2 hours to about 20 hours. In certain embodiments, hybridization of the padlock oligonucleotide to the target nucleic acid can occur at a temperature in the range of about 20°C to about 60°C, e.g., at about 40°C or about 45°C. In certain embodiments, hybridization of the padlock oligonucleotide to the target nucleic acid can occur at a temperature in the range of about 30°C or about 50°C. In certain embodiments, the concentration of the oligonucleotide in the hybridization buffer is about 1 nM to about 1,000 nM, e.g., about 1 nM to about 900 nM, about 1 nM to about 800 nM, about 1 nM to about 700 nM, about 1 nM to about 600 nM, about 1 nM to about 500 nM, about 1 nM to about 400 nM, about 1 nM to about 300 nM, about 1 nM to about 200 nM, about 1 nM to about 100 nM, or about 1 nM to about 50 nM. In certain embodiments, the concentration of the oligonucleotide in the hybridization buffer is about 1 nM to about 50 nM. In certain embodiments, the hybridization buffer can include formamide, e.g., about 10% to about 30% formamide.

[0085] In certain embodiments, ligation using a ligase, such as ligation with SplintR ligase, can be performed after hybridization of the padlock oligonucleotide to the target nucleic acid. In certain embodiments, ligation can occur for a period of about 1 hour to about 24 hours, such as about 1 hour to about 20 hours, about 1 hour to about 16 hours, about 1 hour to about 12 hours, about 5 hours to about 20 hours, or about 5 hours to about 16 hours. In certain embodiments, ligation can occur for a period of about 1 hour to about 24 hours. In certain embodiments, ligation can occur for a period of about 1 hour to about 20 hours. In certain embodiments, ligation can occur for a period of about 1 hour to about 16 hours. In certain embodiments, ligation can occur for a period of about 1 hour to about 12 hours. In certain embodiments, ligation can occur at a temperature in the range of about 10°C to 60°C, such as at 25°C, 30°C, or 40°C. In certain embodiments, ligation can occur at a temperature in the range of about 20°C to 50°C.

[0086] In certain embodiments, multiple oligonucleotides, such as padlock oligonucleotides, can be used in the present disclosure for detecting multiple different target nucleic acids. For example, but not by way of limitation, each oligonucleotide, such as a padlock oligonucleotide, specifically binds to a single target nucleic acid. For example, but not by way of limitation, two or more padlock oligonucleotides, three or more padlock oligonucleotides, four or more padlock oligonucleotides, five or more padlock oligonucleotides, six or more padlock oligonucleotides, seven or more padlock oligonucleotides, eight or more padlock oligonucleotides, nine or more padlock oligonucleotides, or ten or more padlock oligonucleotides can be used in the present disclosure, wherein each padlock oligonucleotide specifically binds to a single target nucleic acid. In certain embodiments, the methods of the present disclosure can include contacting a sample with two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more padlock probes, wherein each padlock oligonucleotide specifically binds to a single target nucleic acid. In certain embodiments, one or more padlock oligonucleotides specifically bind to an oligonucleotide conjugated to a protein binding reagent for detecting a target protein in a sample. In certain embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more padlock probes can each bind to a different oligonucleotide of a protein binding reagent (e.g., multiple protein binding reagents).

[0087] Alternatively and / or additionally, other techniques may be used in the present disclosure for detecting a target nucleic acid. In certain embodiments, other techniques that may be used in the present disclosure for detecting a target nucleic acid include techniques for generating a template (e.g., a circular DNA template) that can be amplified using a polymerase with strand displacement activity (e.g., Phi29 polymerase or a derivative thereof). In certain embodiments, other techniques that may be used in the present disclosure for detecting a target nucleic acid include techniques for amplification using a polymerase with strand displacement activity (e.g., Phi29 polymerase-based amplification). For example, but not by way of limitation, other techniques that may be used in the present disclosure for detecting a target nucleic acid include SNAIL, RCP-FISH, DARTFISH, and OPS. In certain embodiments, SNAIL includes using two oligonucleotides, one of which binds to the target nucleic acid and includes a ligation junction, and a second oligonucleotide that binds to the ligation junction of the target nucleic acid and the first oligonucleotide, followed by ligation to generate a circular DNA molecule. In certain embodiments, DARTFISH includes generating cDNA complementary to the target nucleic acid, followed by using a padlock probe that binds to the cDNA and ligating to generate a circular DNA molecule.

[0088] In certain embodiments, a polymerase with strand displacement activity, such as Phi29 polymerase or a derivative thereof, may then be used to amplify the resulting circularized single-stranded DNA molecule during an RCA process. This RCA process generates single-stranded DNA molecules, referred to herein as "RCA amplicons," that contain tandem repeats of multiple original target nucleic acid sequences. In certain embodiments, the RCA process may be carried out for at least about 1 hour, at least about 5 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours. In certain embodiments, the RCA process may be carried out for at least about 24 hours. In certain embodiments, the RCA process may be carried out for at least about 20 hours. In certain embodiments, the RCA process may be carried out for at least about 16 hours. In certain embodiments, the RCA process may be carried out for at least about 12 hours. In certain embodiments, the RCA process may be carried out for at least about 24 hours. In certain embodiments, the RCA process may be carried out for about 1 to about 24 hours, about 1 to about 22 hours, about 1 to about 20 hours, about 1 to about 18 hours, about 1 to about 16 hours, about 1 to about 14 hours, about 1 to about 12 hours, about 1 to about 10 hours, about 1 to about 8 hours, about 1 to about 6 hours, about 1 to about 4 hours, or about 1 to about 2 hours. In certain embodiments, the RCA process may be carried out for about 8 to about 24 hours. In certain embodiments, the RCA process may be carried out for about 12 to about 24 hours. In certain embodiments, the RCA process may be carried out at a temperature in the range of about 0°C to 50°C, such as at about 30°C.

[0089] In certain embodiments, a sample can be treated with a reagent that prevents degradation of an oligonucleotide conjugated to a protein-binding reagent by blocking the exonuclease activity of a polymerase used in the amplification process. For example, without limitation, a sample can be treated with a reagent that prevents degradation of an oligonucleotide conjugated to a protein-binding reagent by blocking the exonuclease activity, 3'→5' exonuclease activity, of a polymerase (e.g., a polymerase having strand displacement activity, e.g., Phi29 polymerase or a derivative thereof) used in the amplification process (e.g., an RCA process). For example, without limitation, a sample can be treated with a reagent that prevents degradation of the oligonucleotide (e.g., the 3' end of the oligonucleotide of an antibody-oligonucleotide conjugate) of an antibody-oligonucleotide conjugate by blocking the 3'→5' exonuclease activity of Phi29 polymerase or a derivative thereof used in the amplification process (e.g., an RCA process).

[0090] In certain embodiments, a blocking oligonucleotide that binds to an oligonucleotide conjugated to a protein-binding reagent, also referred to herein as a "Phi29 blocking oligonucleotide" or an "exonuclease blocking oligonucleotide", can be used to prevent degradation of the oligonucleotide conjugated to the protein-binding reagent by a polymerase having strand displacement activity (e.g., Phi29 polymerase or a derivative thereof). In certain embodiments, the sample is contacted with the exonuclease blocking oligonucleotide prior to performing the amplification process. In certain embodiments, the sample is contacted with the exonuclease blocking oligonucleotide prior to contacting the sample with a polymerase having strand displacement activity. In certain embodiments, the sample is contacted with the exonuclease blocking oligonucleotide after binding the protein-binding reagent but prior to contacting the sample with a polymerase having strand displacement and / or 3'→5' exonuclease activity. In certain embodiments, the sample is contacted with the exonuclease blocking oligonucleotide prior to contacting the sample with Phi29 polymerase.

[0091] In certain embodiments, the exonuclease blocking oligonucleotide has Figure 4 the structure shown. In certain embodiments, the exonuclease blocking oligonucleotide includes a sequence complementary to the oligonucleotide conjugated to the protein-binding reagent. For example, without limitation, the exonuclease blocking oligonucleotide can include a sequence complementary to a region adjacent to a barcode sequence present in the oligonucleotide conjugated to the protein-binding reagent, as Figure 4 shown. In certain embodiments, this region is 3' of the barcode sequence of the oligonucleotide conjugated to the protein-binding reagent.

[0092] In certain embodiments, the exonuclease blocking oligonucleotide does not bind to the barcode sequence of the oligonucleotide, e.g., as Figure 4As shown. In certain embodiments, the exonuclease-blocking oligonucleotide does not include a nucleotide sequence complementary to the barcode sequence of the oligonucleotide. In certain embodiments, the exonuclease-blocking oligonucleotide does not bind to the primer sequence of the oligonucleotide, e.g., as Figure 4 shown. In certain embodiments, the exonuclease-blocking oligonucleotide does not include a nucleotide sequence complementary to the primer sequence of the oligonucleotide. In certain embodiments, the exonuclease-blocking oligonucleotide includes, at its 3' end, a nucleotide sequence that does not bind to (e.g., is not complementary to) the sequence of the oligonucleotide, e.g., as Figure 4 shown.

[0093] In certain embodiments, the exonuclease-blocking oligonucleotide has a 5' to 3' structure that includes (i) a nucleotide sequence complementary to the oligonucleotide conjugated to the protein-binding reagent (e.g., a first nucleotide sequence, e.g., a complementary region or domain) and (ii) a nucleotide sequence that is not complementary to the oligonucleotide conjugated to the protein-binding reagent (e.g., a second nucleotide sequence, e.g., a 3' flap region or domain). In certain embodiments, the exonuclease-blocking oligonucleotide has a 5' to 3' structure that includes (i) a nucleotide sequence containing an extended template (e.g., a first nucleotide sequence, e.g., a 5' extended template region or domain), (ii) a nucleotide sequence complementary to the oligonucleotide conjugated to the protein-binding reagent (e.g., a second nucleotide sequence, e.g., a complementary region or domain), and (iii) a nucleotide sequence that is not complementary to the oligonucleotide conjugated to the protein-binding reagent (e.g., a third nucleotide sequence, e.g., a 3' flap region or domain), e.g., as Figure 4 shown. In certain embodiments, the nucleotide sequence containing the extended template (e.g., the first nucleotide sequence) is not complementary to the oligonucleotide conjugated to the protein-binding reagent. In certain embodiments, the nucleotide sequence of the oligonucleotide conjugated to the protein-binding reagent bound by the exonuclease-blocking oligonucleotide can be conserved between each of the oligonucleotides conjugated to the various protein-binding reagents used in the methods of the present disclosure.

[0094] In certain embodiments, the exonuclease-blocking oligonucleotide can further include an extension template at its 5' end. In certain embodiments, the extension template at the 5' end of the exonuclease-blocking oligonucleotide can prevent the exonuclease activity of a polymerase (e.g., a polymerase having strand displacement activity, such as Phi29 polymerase). In certain embodiments, the extension template at the 5' end of the exonuclease-blocking oligonucleotide can prevent the 3'→5' exonuclease activity of a polymerase on a single-stranded nucleic acid by generating a double-stranded nucleic acid. In certain embodiments, the extension template at the 5' end of the exonuclease-blocking oligonucleotide can prevent the exonuclease activity of a polymerase (e.g., a polymerase having strand displacement activity, such as, Phi29 polymerase) on the oligonucleotide of a protein-binding reagent-oligonucleotide conjugate. In certain embodiments, the extension template at the 5' end of the exonuclease-blocking oligonucleotide can prevent the exonuclease activity of Phi29 polymerase on the oligonucleotide of a protein-binding reagent-oligonucleotide conjugate.

[0095] In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 5 to about 200 nucleotides, for example, a length of from about 5 to about 150 nucleotides, a length of from about 5 to about 100 nucleotides, a length of from about 5 to about 50 nucleotides, a length of from about 10 to about 150 nucleotides, a length of from about 20 to about 100 nucleotides, or a length of from about 10 to about 100 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 10 to about 100 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 20 to about 90 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 30 to about 90 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 40 to about 90 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 50 to about 90 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 60 to about 90 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 20 to about 80 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 30 to about 80 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 40 to about 80 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 50 to about 80 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 60 to about 80 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 45 to about 75 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 30 to about 60 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide can have a length of from about 5 to about 50 nucleotides, for example, a length of from about 5 to about 45 nucleotides, a length of from about 5 to about 40 nucleotides, a length of from about 5 to about 35 nucleotides, a length of from about 5 to about 30 nucleotides, a length of from about 5 to about 25 nucleotides, a length of from about 5 to about 20 nucleotides, a length of from about 5 to about 15 nucleotides, a length of from about 5 to about 10 nucleotides, a length of from about 10 to about 50 nucleotides, a length of from about 15 to about 50 nucleotides, a length of from about 20 to about 50 nucleotides, a length of from about 25 to about 50 nucleotides, a length of from about 30 to about 50 nucleotides, a length of from about 35 to about 50 nucleotides, a length of from about 40 to about 50 nucleotides, a length of from about 10 to about 40 nucleotides, or a length of from about 10 to about 30 nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide has a length of from about 30 to about 50 nucleotides.

[0096] In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent (e.g., is complementary to the oligonucleotide of the protein-binding reagent) is from about 5 to about 100 nucleotides (e.g., where the nucleotides are contiguous). For example, but not by way of limitation, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent (e.g., is complementary to the oligonucleotide of the protein-binding reagent) is from about 5 to about 95 nucleotides, from about 5 to about 90 nucleotides, from about 5 to about 85 nucleotides, from about 5 to about 80 nucleotides, from about 5 to about 75 nucleotides, from about 5 to about 70 nucleotides, from about 5 to about 65 nucleotides, from about 5 to about 60 nucleotides, from about 5 to about 55 nucleotides, from about 5 to about 50 nucleotides, from about 5 to about 45 nucleotides, from about 5 to about 40 nucleotides, from about 5 to about 35 nucleotides, from about 5 to about 30 nucleotides, from about 5 to about 25 nucleotides, from about 5 to about 20 nucleotides, from about 5 to about 15 nucleotides, from about 5 to about 10 nucleotides, from about 10 to about 100 nucleotides, from about 15 to about 100 nucleotides, from about 20 to about 100 nucleotides, from about 25 to about 100 nucleotides, from about 30 to about 100 nucleotides, from about 35 to about 100 nucleotides, from about 40 to about 100 nucleotides, from about 45 to about 100 nucleotides, from about 50 to about 100 nucleotides, from about 55 to about 100 nucleotides, from about 60 to about 100 nucleotides, from about 65 to about 100 nucleotides, from about 70 to about 100 nucleotides, from about 75 to about 100 nucleotides, from about 80 to about 100 nucleotides, from about 85 to about 100 nucleotides, from about 90 to about 100 nucleotides, from about 95 to about 100 nucleotides, from about 30 to about 90 nucleotides, from about 30 to about 80 nucleotides, from about 30 to about 70 nucleotides, from about 30 to about 60 nucleotides, from about 30 to about 50 nucleotides, from about 10 to about 40 nucleotides or from about 10 to about 30 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent (e.g., is complementary to the oligonucleotide of the protein-binding reagent) is from about 5 to about 50 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent (e.g., is complementary to the oligonucleotide of the protein-binding reagent) is from about 5 to about 40 nucleotides.In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent (e.g., is complementary to the oligonucleotide of the protein-binding reagent) is from about 5 to about 30 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent (e.g., is complementary to the oligonucleotide of the protein-binding reagent) is from about 10 to about 50 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent (e.g., is complementary to the oligonucleotide of the protein-binding reagent) is from about 10 to about 40 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent (e.g., is complementary to the oligonucleotide of the protein-binding reagent) is from about 20 to about 30 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is from about 50 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is from about 60 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is from about 70 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is from about 80 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is from about 90 to about 100 nucleotides.

[0097] In certain embodiments, the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% complementary to the nucleotide sequence of the oligonucleotide of the protein-binding reagent. In certain embodiments, the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is at least about 90% complementary to the nucleotide sequence of the oligonucleotide of the protein-binding reagent. In certain embodiments, the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is at least about 95% complementary to the nucleotide sequence of the oligonucleotide of the protein-binding reagent. In certain embodiments, the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is at least about 97% complementary to the nucleotide sequence of the oligonucleotide of the protein-binding reagent. In certain embodiments, the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is at least about 98% complementary to the nucleotide sequence of the oligonucleotide of the protein-binding reagent. In certain embodiments, the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is at least about 99% complementary to the nucleotide sequence of the oligonucleotide of the protein-binding reagent. In certain embodiments, the nucleotide sequence of the exonuclease-blocking oligonucleotide that binds to the oligonucleotide of the protein-binding reagent is at least about 100% complementary to the nucleotide sequence of the oligonucleotide of the protein-binding reagent.

[0098] In certain embodiments, about 10% to about 90% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. For example, but not by way of limitation, about 15% to about 90%, about 20% to about 90%, about 25% to about 90%, about 30% to about 90%, about 35% to about 90%, about 40% to about 45%, about 50% to about 90%, about 55% to about 90%, about 60% to about 90%, about 65% to about 90%, about 70% to about 90%, about 80% to about 90%, about 85% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 80% or about 60% to about 70% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, about 50% to about 90% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, about 60% to about 90% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, about 70% to about 90% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, about 80% to about 90% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, about 20% to about 60% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, about 10% to about 50% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, about 20% to about 60% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, about 30% to about 60% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, about 40% to about 60% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent.In certain embodiments, from about 50% to about 60% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, from about 20% to about 50% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, from about 30% to about 50% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, from about 40% to about 50% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent. In certain embodiments, from about 30% to about 40% of the nucleotide sequence of the exonuclease-blocking oligonucleotide is complementary to the oligonucleotide of the protein-binding reagent.

[0099] In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 5 to about 100 nucleotides (e.g., where the nucleotides are contiguous). For example, but not by way of limitation, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 5 to about 95 nucleotides, from about 5 to about 90 nucleotides, from about 5 to about 85 nucleotides, from about 5 to about 80 nucleotides, from about 5 to about 75 nucleotides, from about 5 to about 70 nucleotides, from about 5 to about 65 nucleotides, from about 5 to about 60 nucleotides, from about 5 to about 55 nucleotides, from about 5 to about 50 nucleotides, from about 5 to about 45 nucleotides, from about 5 to about 40 nucleotides, from about 5 to about 35 nucleotides, from about 5 to about 30 nucleotides, from about 5 to about 25 nucleotides, from about 5 to about 20 nucleotides, from about 5 to about 15 nucleotides, from about 5 to about 10 nucleotides, from about 10 to about 100 nucleotides, from about 15 to about 100 nucleotides, from about 20 to about 100 nucleotides, from about 25 to about 100 nucleotides, from about 30 to about 100 nucleotides, from about 35 to about 100 nucleotides, from about 40 to about 100 nucleotides, from about 45 to about 100 nucleotides, from about 50 to about 100 nucleotides, from about 55 to about 100 nucleotides, from about 60 to about 100 nucleotides, from about 65 to about 100 nucleotides, from about 70 to about 100 nucleotides, from about 75 to about 100 nucleotides, from about 80 to about 100 nucleotides, from about 85 to about 100 nucleotides, from about 90 to about 100 nucleotides, from about 95 to about 100 nucleotides, from about 30 to about 90 nucleotides, from about 30 to about 80 nucleotides, from about 30 to about 70 nucleotides, from about 30 to about 60 nucleotides, from about 30 to about 50 nucleotides, from about 10 to about 40 nucleotides or from about 10 to about 30 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 50 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 60 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 70 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 80 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 90 to about 100 nucleotides.In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 20 to about 60 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 20 to about 50 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 20 to about 40 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 20 to about 30 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 10 to about 40 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 10 to about 30 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 10 to about 20 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide comprising the extension template is from about 20 to about 30 nucleotides.

[0100] In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent (e.g., is not complementary to the oligonucleotide of the protein-binding reagent) is from about 5 to about 100 nucleotides (e.g., where the nucleotides are contiguous). In certain embodiments, the length of the nucleotide sequence at the 3'-end of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent (e.g., is not complementary to the oligonucleotide of the protein-binding reagent) is from about 5 to about 100 nucleotides (e.g., where the nucleotides are contiguous). For example, but not by way of limitation, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent (e.g., is not complementary to the oligonucleotide of the protein-binding reagent) is from about 5 to about 95 nucleotides, from about 5 to about 90 nucleotides, from about 5 to about 85 nucleotides, from about 5 to about 80 nucleotides, from about 5 to about 75 nucleotides, from about 5 to about 70 nucleotides, from about 5 to about 65 nucleotides, from about 5 to about 60 nucleotides, from about 5 to about 55 nucleotides, from about 5 to about 50 nucleotides, from about 5 to about 45 nucleotides, from about 5 to about 40 nucleotides, from about 5 to about 35 nucleotides, from about 5 to about 30 nucleotides, from about 5 to about 25 nucleotides, from about 5 to about 20 nucleotides, from about 5 to about 15 nucleotides, from about 5 to about 10 nucleotides, from about 10 to about 100 nucleotides, from about 15 to about 100 nucleotides, from about 20 to about 100 nucleotides, from about 25 to about 100 nucleotides, from about 30 to about 100 nucleotides, from about 35 to about 100 nucleotides, from about 40 to about 100 nucleotides, from about 45 to about 100 nucleotides, from about 50 to about 100 nucleotides, from about 55 to about 100 nucleotides, from about 60 to about 100 nucleotides, from about 65 to about 100 nucleotides, from about 70 to about 100 nucleotides, from about 75 to about 100 nucleotides, from about 80 to about 100 nucleotides, from about 85 to about 100 nucleotides, from about 90 to about 100 nucleotides, from about 95 to about 100 nucleotides, from about 30 to about 90 nucleotides, from about 30 to about 80 nucleotides, from about 30 to about 70 nucleotides, from about 30 to about 60 nucleotides, from about 30 to about 50 nucleotides, from about 10 to about 40 nucleotides or from about 10 to about 30 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 50 to about 100 nucleotides.In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 60 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 70 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 80 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 90 to about 100 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 20 to about 80 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 20 to about 60 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 20 to about 50 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 25 to about 45 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide that does not bind to the oligonucleotide of the protein-binding reagent is from about 5 to about 40 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide (e.g., at the 3' end) that does not bind to the oligonucleotide of the protein-binding reagent is from about 5 to about 35 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide (e.g., at the 3' end) that does not bind to the oligonucleotide of the protein-binding reagent is from about 5 to about 30 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide (e.g., at the 3' end) that does not bind to the oligonucleotide of the protein-binding reagent is from about 5 to about 25 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide (e.g., at the 3' end) that does not bind to the oligonucleotide of the protein-binding reagent is from about 5 to about 20 nucleotides. In certain embodiments, the length of the nucleotide sequence of the exonuclease-blocking oligonucleotide (e.g., at the 3' end) that does not bind to the oligonucleotide of the protein-binding reagent is from about 10 to about 20 nucleotides.

[0101] In certain embodiments, the exonuclease-blocking oligonucleotide may include one or more modified nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide includes at least 5 or more, at least 6 or more, at least 7 or more, at least 8 or more, at least 9 or more, at least 10 or more, at least 11 or more, at least 12 or more, at least 13 or more, at least 14 or more, at least 15 or more, at least 16 or more, at least 17 or more, at least 18 or more, at least 19 or more, or at least 20 or more modified nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide includes from about 5 to about 20 modified nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide includes from about 5 to about 15 modified nucleotides. In certain embodiments, the exonuclease-blocking oligonucleotide includes from about 10 to about 15 modified nucleotides. In certain embodiments, the modified nucleotide is a nucleotide that is resistant to exonuclease cleavage. In certain embodiments, the modified nucleotide includes a modified nucleobase having a derivatized sugar or phosphate backbone bond or a chemically modified residue. In certain embodiments, the modified nucleotide is a nucleotide having a phosphate backbone modification. In certain embodiments, the modified nucleotide is a nucleotide having a phosphorothioate bond at its 3'-end. In certain embodiments, the modified nucleotide is a 2',3'-dideoxynucleoside-α-thiol nucleotide, e.g., 2',3'-dideoxyadenosine-5'-O-(1-thiotriphosphate), 2',3'-dideoxycytidine-5'-O-(1-thiotriphosphate), 2',3'-dideoxyguanosine-5'-O-(1-thiotriphosphate), and / or 2',3'-dideoxythymidine-5'-O-(1-thiotriphosphate). In certain embodiments, the exonuclease-blocking oligonucleotide includes at least 5 or more, at least 6 or more, at least 7 or more, at least 8 or more, at least 9 or more, at least 10 or more, at least 11 or more, at least 12 or more, at least 13 or more, at least 14 or more, at least 15 or more, at least 16 or more, at least 17 or more, at least 18 or more, at least 19 or more, or at least 20 or more nucleotides having a phosphorothioate bond at the 3'-end of the exonuclease-blocking oligonucleotide that does not bind (e.g., is not complementary to) the barcode sequence of the oligonucleotide, e.g., at the 3'-end. In certain embodiments, the exonuclease-blocking oligonucleotide includes at least 10 or more modified nucleotides at the 3'-end, e.g., nucleotides having a phosphate backbone modification, e.g., nucleotides having a phosphorothioate bond. In certain embodiments, the modified nucleotides (e.g., nucleotides containing a phosphorothioate bond) are contiguous.The use of phosphorothioate linkages can prevent DNA barcodes from becoming double-stranded (e.g., upon contact with exonuclease-blocking oligonucleotides). In certain embodiments, the sample is contacted with an exonuclease-blocking oligonucleotide prior to performing the amplification process.

[0102] In certain embodiments, the sample can be contacted with an exonuclease-blocking oligonucleotide for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, or about 30 minutes or less. In certain embodiments, the sample can be contacted with an exonuclease-blocking oligonucleotide for about 30 minutes.

[0103] In certain embodiments, one or more of the padlock oligonucleotides can hybridize to a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that is complementary to an oligonucleotide conjugated to a protein-binding reagent. In certain embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more padlock oligonucleotides can each bind to a different blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide). In certain embodiments, each different blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) hybridizes to an oligonucleotide conjugated to a protein-binding reagent.

[0104] In certain embodiments, one or more of the plurality of padlock oligonucleotides can hybridize to a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that is complementary to an oligonucleotide conjugated to a protein-binding reagent, and a second padlock oligonucleotide of the plurality of padlock oligonucleotides binds to a target nucleic acid (e.g., RNA or genomic nucleic acid of the sample).

[0105] In certain embodiments, a sample processed according to the methods disclosed herein can be permeabilized a second time, e.g., before performing an amplification process and after contacting the sample with a protein-binding reagent. For example, a permeabilization reagent disclosed herein can be used to permeabilize such a sample a second time. In certain embodiments, permeabilization is performed using an acid. For example, but not by way of limitation, HCl (e.g., from about 0.01 N to about 10 N HCl or from about 0.01 N to about 1.0 N HCl) can be used for permeabilization. In certain embodiments, a solution comprising an acid (e.g., HCl) and a peptidase can be used for permeabilization. In certain embodiments, the peptidase can be pepsin. In certain embodiments, the peptidase (e.g., pepsin) can be included in the solution at a concentration of from about 0.1 mg / ml to about 10 mg / ml or from about 0.1 mg / ml to about 5 mg / ml. In certain embodiments, a solution comprising from about 0.01 N to about 1 N HCl and from about 0.1 mg / ml to about 5 mg / ml peptidase (e.g., pepsin) can be used for permeabilization. In certain embodiments, the sample can be contacted with HCl for about 1 to about 10 minutes.

[0106] In certain embodiments, prior to an amplification process for nucleic acid detection, a sample processed according to the methods disclosed herein is treated with NHS-acetic acid. For example, but not by way of limitation, the sample can be treated with NHS-acetic acid after post-fixation of the sample and HCl permeabilization. In certain embodiments, the sample can be contacted with NHS-acetic acid for about 5 hours or less, about 4 hours or less, about 3 hours or less, about 2 hours or less, about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, or about 30 minutes or less. In certain embodiments, the sample can be contacted with NHS-acetic acid for about 30 minutes.

[0107] D. Imaging Strategies

[0108] As described above, the methods of the present disclosure include imaging target proteins and target nucleic acids in a sample. In certain embodiments, oligonucleotides conjugated to protein-binding reagents and / or amplicons generated by an amplification process facilitate imaging of the target proteins and nucleic acids.

[0109] In certain embodiments, detection probes can be used to image oligonucleotides that are bound to protein binding reagents and / or amplicons used in the methods disclosed herein. A "detection probe" refers to an oligonucleotide that can selectively hybridize under appropriate hybridization conditions to at least a portion of a target sequence (e.g., a portion of a target sequence that has been amplified during RCA or a portion of an oligonucleotide conjugated to a protein binding reagent). In certain embodiments, the detection probe can comprise or consist of from about 10 to about 50 nucleotides (e.g., from about 15 to about 30 nucleotides). In certain embodiments, the detection probe used in the present disclosure comprises a sequence that specifically hybridizes to an amplicon (e.g., an RCA amplicon). In certain embodiments, the detection probe used in the present disclosure comprises a sequence that specifically hybridizes to the DNA barcode sequence of an oligonucleotide conjugated to a protein binding reagent (e.g., an antibody). In certain embodiments, the detection probe used in the present disclosure comprises a sequence that specifically hybridizes to a detection bridge oligonucleotide.

[0110] In certain embodiments, the detection bridge oligonucleotide for protein imaging is an oligonucleotide that hybridizes to the nucleotide sequence (e.g., the DNA barcode sequence) of an oligonucleotide conjugated to a protein binding reagent (e.g., an antibody). In certain embodiments, the detection bridge oligonucleotide can comprise a sequence complementary to the DNA barcode and additional nucleotides present in the oligonucleotide conjugated to the protein binding reagent (e.g., antibody), as Figure 1 shown. For example, but not by way of limitation, the detection bridge oligonucleotide can comprise a sequence complementary to the DNA barcode and at least five additional nucleotides present in the oligonucleotide conjugated to the protein binding reagent (e.g., antibody). In certain embodiments, the detection bridge oligonucleotide comprises a nucleotide sequence having a length of from about 5 to about 100 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., antibody). In certain embodiments, where the method comprises amplifying an exonuclease-blocking oligonucleotide and / or an oligonucleotide conjugated to a protein binding reagent, the detection bridge oligonucleotide can comprise a sequence complementary to the nucleotide sequence of the resulting amplicon. In certain embodiments, the detection bridge oligonucleotide comprises a nucleotide sequence having a length of from about 5 to about 100 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the resulting amplicon.

[0111] In certain embodiments, the detection bridge oligonucleotide for nucleic acid imaging is an oligonucleotide that hybridizes to a sequence present in an amplicon generated during an amplification reaction, e.g., as Figure 1As shown. In certain embodiments, the padlock oligonucleotide includes such sequences that are then amplified during an amplification process (such as RCA). In certain embodiments, the amplicon includes one or more, two or more, three or more, four or more, or five or more sequences that can hybridize to a detection bridge oligonucleotide.

[0112] In certain embodiments, the detection bridge oligonucleotide includes two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more nucleotide sequences that can hybridize to a detection probe. In certain embodiments, the detection bridge oligonucleotide includes five or more nucleotide sequences that can hybridize to a detection probe. In certain embodiments, the detection bridge oligonucleotide includes ten or more nucleotide sequences that can hybridize to a detection probe. In certain embodiments, the detection bridge oligonucleotide includes from about one to about eight nucleotide sequences that can hybridize to a detection probe. In certain embodiments, one or more nucleotide sequences that can hybridize to a detection probe are the same.

[0113] In certain embodiments, the detection bridge oligonucleotide has a length of about 5 to about 200 nucleotides, for example, a length of about 5 to about 150 nucleotides, a length of about 5 to about 100 nucleotides, a length of about 5 to about 50 nucleotides, a length of about 10 to about 150 nucleotides, a length of about 20 to about 100 nucleotides, or a length of about 10 to about 100 nucleotides. In certain embodiments, the detection bridge oligonucleotide has a length of about 25 to about 100 nucleotides or a length of about 25 to about 120 nucleotides. In certain embodiments, the detection bridge oligonucleotide has a length of about 25 to about 100 nucleotides. In certain embodiments, the detection bridge oligonucleotide has a length of about 25 to about 120 nucleotides. In certain embodiments, the detection bridge oligonucleotide includes one or more nucleotide sequences that can hybridize to a detection probe. In certain embodiments, the detection bridge oligonucleotide includes from about one to about eight nucleotide sequences that can hybridize to a detection probe. In certain embodiments, the detection bridge oligonucleotide includes from about one to about ten nucleotide sequences that can hybridize to a detection probe.

[0114] In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence having a length of from about 5 to about 100 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon. In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence having a length of from about 5 to about 50 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon. In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence having a length of from about 10 to about 40 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon. In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence having a length of from about 10 to about 30 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon. In certain embodiments, the detection bridging oligonucleotide comprises a nucleotide sequence having a length of from about 10 to about 20 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon.

[0115] In certain embodiments, the detection bridging oligonucleotide for detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of from about 5 to about 40 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon obtained from amplifying the target nucleic acid. In certain embodiments, the detection bridging oligonucleotide for detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of from about 5 to about 35 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon obtained from amplifying the target nucleic acid. In certain embodiments, the detection bridging oligonucleotide for detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of from about 5 to about 30 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon obtained from amplifying the target nucleic acid. In certain embodiments, the detection bridging oligonucleotide for detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of from about 5 to about 25 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon obtained from amplifying the target nucleic acid. In certain embodiments, the detection bridging oligonucleotide for detecting a target nucleic acid (e.g., mRNA) comprises a nucleotide sequence having a length of from about 5 to about 20 nucleotides (e.g., wherein the nucleotides are contiguous) that is complementary to the nucleotide sequence of the amplicon obtained from amplifying the target nucleic acid.

[0116] In certain embodiments, a detection bridge oligonucleotide for detecting an oligonucleotide conjugated to a protein binding reagent includes a nucleotide sequence having from about 5 to about 50 nucleotides (e.g., where the nucleotides are contiguous) that is complementary to the oligonucleotide conjugated to the protein binding reagent. In certain embodiments, a detection bridge oligonucleotide for detecting an oligonucleotide conjugated to a protein binding reagent includes a nucleotide sequence having from about 5 to about 40 nucleotides (e.g., where the nucleotides are contiguous) that is complementary to the oligonucleotide conjugated to the protein binding reagent. In certain embodiments, a detection bridge oligonucleotide for detecting an oligonucleotide conjugated to a protein binding reagent includes a nucleotide sequence having from about 5 to about 35 nucleotides (e.g., where the nucleotides are contiguous) that is complementary to the oligonucleotide conjugated to the protein binding reagent. In certain embodiments, a detection bridge oligonucleotide for detecting an oligonucleotide conjugated to a protein binding reagent includes a nucleotide sequence having from about 5 to about 30 nucleotides (e.g., where the nucleotides are contiguous) that is complementary to the oligonucleotide conjugated to the protein binding reagent. In certain embodiments, a detection bridge oligonucleotide for detecting an oligonucleotide conjugated to a protein binding reagent includes a nucleotide sequence having from about 10 to about 30 nucleotides (e.g., where the nucleotides are contiguous) that is complementary to the oligonucleotide conjugated to the protein binding reagent. In certain embodiments, a detection bridge oligonucleotide for detecting an oligonucleotide conjugated to a protein binding reagent includes a nucleotide sequence having from about 20 to about 30 nucleotides (e.g., where the nucleotides are contiguous) that is complementary to the oligonucleotide conjugated to the protein binding reagent.

[0117] In certain embodiments, the detection probe is conjugated to a detectable label to facilitate imaging. Non-limiting examples of detectable labels include fluorescent labels (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.), rhodamine, phycobiliproteins and R-phycoerythrin, and quantum dots (e.g., cadmium selenide capped with zinc sulfide)), chromogenic labels, electron-dense labels, chemiluminescent labels, and radiolabels. In certain embodiments, the detection probe is fluorescently labeled. In certain embodiments, the detection probe is covalently bound to a fluorescent label at its 5' end or 3' end.

[0118] In certain embodiments, the detection bridge oligonucleotide can include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more detectable labels described herein, which can obviate the need for a detection probe.

[0119] In certain embodiments, where multiple target nucleic acids are to be imaged in a single sample, specific detection probes, such as detection probes for specific target nucleic acids, can each be labeled with a different label (e.g., a fluorophore), thereby allowing simultaneous imaging of multiple target nucleic acids. Similarly, in certain embodiments, where multiple target nucleic acids are to be imaged in a single sample, specific detection probes, such as detection probes for specific target nucleic acids, can each be labeled with a different label (e.g., a fluorophore), thereby allowing simultaneous imaging of multiple target nucleic acids.

[0120] In certain embodiments, cycling imaging is performed to visualize labeled target proteins and target nucleic acids in a single sample. In certain embodiments, cycling imaging involves the cyclic addition and removal of labeled detection probes. In certain embodiments, removal of the labeled detection probe is achieved by heat denaturation. Alternatively or additionally, removal of the labeled detection probe is achieved by using a detection probe with a disulfide-conjugated dye, thereby allowing cleavage of the disulfide to remove the label from the detection probe.

[0121] In certain embodiments, cycling imaging is first performed on all protein targets and then on all nucleic acid targets. Alternatively, cycling imaging is first performed on all nucleic acid targets and then on all protein targets. In certain embodiments, a first target protein is imaged, followed by a first target nucleic acid, and the process is repeated until all protein and nucleic acid targets are imaged.

[0122] In certain embodiments, cycling imaging can be used to detect target proteins in a sample. For example, but not by way of limitation, cycling imaging can be performed by contacting the sample with a detection probe (e.g., a fluorescently labeled detection probe) that is specific for the barcode sequence of an oligonucleotide conjugated to an antibody that binds to a target sequence. In certain embodiments, the detection probe is then imaged and subsequently removed. In certain embodiments, the detection probe is removed by a chaotropic solvent. In certain embodiments, one or more new detection probes are bound and then imaged. In certain embodiments, the cycling imaging process is repeated until all antibodies that bind to the target proteins in the sample have been imaged.

[0123] In certain embodiments, cyclic imaging of a target nucleic acid can be performed. For example, but not by way of limitation, cyclic imaging can be performed by contacting the sample with a detection probe (e.g., a fluorescently labeled detection probe) that is specific for an amplicon (e.g., an RCA amplicon) generated during amplification of the target nucleic acid. In certain embodiments, the detection probe is then imaged and subsequently removed, for example, by a chaotropic solvent and / or by a thermal process. In certain embodiments, one or more new detection probes are bound and subsequently imaged. In certain embodiments, the cyclic imaging process is repeated until all of the target nucleic acids in the sample have been imaged.

[0124] Additional disclosure regarding cyclic imaging is provided in Black et al., Nature Protocols 16:3802-3835 (2021) and Kennedy-Darling et al., Eur. J. Immunol. 51(5):1262-1277 (2021), the contents of each of which are incorporated herein by reference in their entirety.

[0125] In certain embodiments, methods for imaging target proteins and target nucleic acids in a sample include: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to a target protein in the sample, wherein the protein-binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that includes a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (d) amplifying the target nucleic acid in the sample by performing an amplification process to generate an amplicon; (e) contacting the sample with a first labeled detection probe that includes a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second labeled detection probe that includes a sequence complementary to the sequence of the amplicon; and (h) imaging the second labeled detection probe to detect the target nucleic acid. In certain embodiments, the target nucleic acid is the blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) and / or the target nucleic acid is the oligonucleotide conjugated to the protein-binding reagent.

[0126] In certain embodiments, methods for imaging a target protein and a target nucleic acid in a sample include: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to the target protein in the sample, wherein the protein-binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that includes a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (d) amplifying the target nucleic acid in the sample by performing an amplification process to produce amplicons; (e) contacting the sample with a first labeled detection probe that includes a sequence complementary to the sequence of the amplicons; (f) imaging the first labeled detection probe to detect the target nucleic acid; (g) contacting the sample with a second labeled detection probe that includes a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; and (h) imaging the second labeled detection probe to detect the target protein. In certain embodiments, the target nucleic acid is the blocking oligonucleotide and / or the target nucleic acid is the oligonucleotide conjugated to the protein-binding reagent.

[0127] In certain embodiments, methods for imaging a target protein and a target nucleic acid in a sample include: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to the target protein in the sample, wherein the protein-binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that includes a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (d) amplifying the blocking oligonucleotide and / or the oligonucleotide conjugated to the protein-binding reagent in the sample by performing an amplification process to produce a first amplicon; (e) amplifying the target nucleic acid in the sample by performing an amplification process to produce a second amplicon; (f) contacting the sample with a first labeled detection probe that includes a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent, and / or contacting the sample with a first labeled detection probe that includes a sequence complementary to the first amplicon; (g) imaging the first labeled detection probe to detect the target protein; (h) contacting the sample with a second labeled detection probe that includes a sequence complementary to the sequence of the second amplicon; and (i) imaging the second labeled detection probe to detect the target nucleic acid. In certain embodiments, the production of the first amplicon and the second amplicon occurs simultaneously during a single amplification process. In certain embodiments, the production of the first amplicon occurs during a first amplification process, and the second amplicon occurs during a second amplification process, which are performed separately.

[0128] In certain embodiments, methods for imaging a target protein and a target nucleic acid in a sample include: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to the target protein in the sample, wherein the protein-binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (d) amplifying the blocking oligonucleotide and / or the oligonucleotide conjugated to the protein-binding reagent by performing an amplification process to produce a first amplicon; (e) amplifying the target nucleic acid in the sample by performing an amplification process to produce a second amplicon; (f) contacting the sample with a first labeled detection probe that comprises a sequence complementary to the sequence of the second amplicon; (g) imaging the first labeled detection probe to detect the target nucleic acid; (h) contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent, and / or contacting the sample with a second labeled detection probe that comprises a sequence complementary to the first amplicon; and (i) imaging the second labeled detection probe to detect the target protein. In certain embodiments, the production of the first amplicon and the second amplicon occurs simultaneously during a single amplification process. In certain embodiments, the production of the first amplicon occurs during a first amplification process and the second amplicon occurs during a second amplification process, which are performed separately.

[0129] In certain embodiments, methods for imaging a target protein and a target nucleic acid in a sample include: (a) binding a protein-binding reagent conjugated to an oligonucleotide to the target protein in the sample; (b) hybridizing a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) to the oligonucleotide conjugated to the protein-binding reagent; (c) amplifying the target nucleic acid in the sample by performing an amplification process to produce an amplicon; (d) imaging the target protein by detecting a first labeled detection probe that hybridizes to the oligonucleotide conjugated to the protein-binding reagent; and (e) imaging the target nucleic acid by detecting a second labeled detection probe that hybridizes to the amplicon. In certain embodiments, the target nucleic acid is the blocking oligonucleotide and / or the target nucleic acid is the oligonucleotide conjugated to the protein-binding reagent.

[0130] In certain embodiments, methods for imaging a target protein and a target nucleic acid in a sample include: (a) binding a protein binding reagent conjugated to an oligonucleotide to the target protein in the sample; (b) hybridizing a blocking oligonucleotide to the oligonucleotide conjugated to the protein binding reagent; (c) amplifying the target nucleic acid in the sample by performing an amplification process to produce amplicons; (d) imaging the target nucleic acid by detecting a first labeled detection probe that hybridizes to the amplicons; and (e) imaging the target protein by detecting a second labeled detection probe that hybridizes to the oligonucleotide conjugated to the protein binding reagent. In certain embodiments, the target nucleic acid is the blocking oligonucleotide and / or the target nucleic acid is the oligonucleotide conjugated to the protein binding reagent.

[0131] In certain embodiments, methods for imaging a target protein and a target nucleic acid in a sample include: (a) binding a protein binding reagent conjugated to an oligonucleotide to the target protein in the sample; (b) hybridizing a blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) to the oligonucleotide conjugated to the protein binding reagent; (c) amplifying the blocking oligonucleotide and / or the oligonucleotide conjugated to the protein binding reagent in the sample by performing an amplification process to produce a first amplicon; (d) amplifying the target nucleic acid in the sample by performing an amplification process to produce a second amplicon; (e) imaging the target protein by detecting a first labeled detection probe that hybridizes to the oligonucleotide conjugated to the protein binding reagent and / or to the first amplicon; and (f) imaging the target nucleic acid by detecting a second labeled detection probe that hybridizes to the second amplicon. In certain embodiments, the production of the first amplicon and the second amplicon occurs simultaneously during a single amplification process. In certain embodiments, the production of the first amplicon occurs during a first amplification process and the second amplicon occurs during a second amplification process, which are performed separately.

[0132] In certain embodiments, methods for imaging a target protein and a target nucleic acid in a sample include: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to the target protein in the sample, wherein the protein-binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (d) amplifying the target nucleic acid in the sample by performing an amplification process to produce an amplicon; (e) contacting the sample with a first bridging oligonucleotide that comprises a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent, and contacting the sample with a first labeled detection probe that comprises a sequence complementary to the sequence of the first bridging oligonucleotide; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second bridging oligonucleotide that comprises a sequence complementary to the sequence of the amplicon, and contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the second bridging oligonucleotide; and (h) imaging the second labeled detection probe to detect the target nucleic acid.

[0133] In certain embodiments, methods for imaging a target protein and a target nucleic acid in a sample include: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to the target protein in the sample, wherein the protein-binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease-blocking oligonucleotide) that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (d) amplifying the target nucleic acid in the sample by performing an amplification process to produce an amplicon; (e) contacting the sample with a first bridging oligonucleotide that comprises a sequence complementary to the sequence of the amplicon, and contacting the sample with a first labeled detection probe that comprises the sequence of the amplicon; (f) imaging the first labeled detection probe to detect the target nucleic acid; (g) contacting the sample with a second bridging oligonucleotide that comprises a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent, and contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the second bridging oligonucleotide; and (h) imaging the second labeled detection probe to detect the target protein.

[0134] III. Systems and Kits

[0135] The present disclosure provides systems and kits for performing the methods of the present disclosure. For example, but not by way of limitation, the present disclosure provides systems and kits containing materials for performing methods for imaging nucleic acids and proteins in a sample.

[0136] In certain embodiments, the systems or kits of the present disclosure include a container containing one or more protein-binding reagents. In certain embodiments, the systems or kits may further include a container containing one or more blocking oligonucleotides. In certain embodiments, the systems or kits of the present disclosure may further include one or more detection probes, such as fluorescently labeled detection probes. Non-limiting examples of suitable containers include bottles, test tubes, vials, and microtiter plates. The containers can be formed from a variety of materials, such as glass or plastic.

[0137] In certain embodiments, the systems or kits further include a packaging insert that provides instructions for using the components provided in the systems or kits. For example, the systems or kits of the present disclosure may include a packaging insert that provides instructions for performing methods for imaging one or more target proteins and one or more target nucleic acids in a single sample.

[0138] In certain embodiments, the systems or kits of the present disclosure may further include reagents for performing an amplification reaction (e.g., an RCA reaction). In certain embodiments, the reagents may include one or more of the following: polymerase, reverse transcriptase, nucleoside triphosphates or NTP analogs, primers, cofactors, ligation reaction reagents, endonucleases, lysis reagents, dyes, markers, RNase inhibitors, and labels.

[0139] In certain embodiments, from a commercial and user perspective, the systems or kits may include other desired materials, including other buffers and diluents. In certain embodiments, the systems or kits may include materials or reagents for permeabilizing, fixing, and / or crosslinking cells or cell nuclei. In certain embodiments, the systems or kits of the present disclosure may include a fixative, such as formaldehyde. In certain embodiments, the systems or kits may include NHS-acetic acid. In certain embodiments, the systems or kits may include HCl.

[0140] In certain embodiments, the components of the systems or kits are provided in a predetermined ratio, and the relative amounts of the various reagents are appropriately varied to obtain the desired sensitivity and throughput of the disclosed methods.

[0141] IV. Exemplary Embodiments

[0142] A. The present disclosure provides a method for imaging target proteins and target nucleic acids in a sample, the method comprising: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to a target protein in the sample, wherein the protein-binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (d) amplifying the target nucleic acid in the sample by performing an amplification process to generate amplicons; (e) contacting the sample with a first labeled detection probe that comprises a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the amplicon; and (h) imaging the second labeled detection probe to detect the target nucleic acid.

[0143] A1. The method according to A, wherein the amplification process is a rolling circle amplification process.

[0144] A2. The method according to A1, wherein the rolling circle amplification process comprises: (a) contacting the sample with (i) a padlock probe comprising two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to generate a circular DNA template; and (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

[0145] A3. The method according to any one of A to A2, wherein the protein-binding reagent is an antibody or an antigen-binding fragment thereof.

[0146] A4. The method according to any one of A to A3, wherein the target nucleic acid comprises RNA.

[0147] A5. The method according to any one of A to A4, wherein providing the sample comprises one or more of the following: (a) treating the sample with a fixative; (b) dehydrating the sample; and (c) permeabilizing the sample.

[0148] A6. The method according to any one of A to A5, wherein at least 10 target proteins are imaged in the sample.

[0149] A7. The method according to any one of A to A6, wherein at least 10 target nucleic acids are imaged in the sample.

[0150] A8. The method according to any one of A to A7, wherein the sample is post-fixed before amplifying the target nucleic acid.

[0151] A9. The method according to any one of A to A8, wherein the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid.

[0152] A10. The method according to any one of A to A9, wherein the sample is treated with NHS-acetic acid before amplifying the target nucleic acid.

[0153] A11. The method according to any one of A to A10, wherein the sample is a tissue sample.

[0154] A12. The method according to any one of A to A11, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent is located at the 5'-end of the blocking oligonucleotide.

[0155] A13. The method according to any one of A to A12, wherein the oligonucleotide conjugated to the protein binding reagent comprises a barcode sequence, and wherein the blocking oligonucleotide does not bind to the barcode sequence.

[0156] A14. The method according to any one of A to A13, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

[0157] A15. The method according to A14, wherein one or more modified nucleotides are located at the 3'-end of the blocking oligonucleotide, for example, within the 3'-flap region.

[0158] A16. The method according to any one of A to A13, wherein the blocking oligonucleotide comprises at least about 10 modified nucleotides.

[0159] A17. The method according to A16, wherein at least about 10 modified nucleotides are located at the 3'-end of the blocking oligonucleotide, for example, within the 3'-flap region.

[0160] A18. The method according to any one of A14 to A17, wherein one or more modified nucleotides and / or wherein at least 10 modified nucleotides comprise nucleotides having a phosphodiester backbone modification (e.g., phosphorothioate bond).

[0161] A19. The method according to any one of A to A18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent has a length of about 50 to about 100 nucleotides.

[0162] A20. The method according to any one of A to A18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent has a length of about 10 to about 40 nucleotides.

[0163] A21. The method according to any one of A to A18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent has a length of about 20 to about 30 nucleotides.

[0164] A22. The method according to A19, A20 or A22, wherein about 50 to about 100 nucleotides, about 10 to about 40 nucleotides and / or about 20 to about 30 nucleotides are consecutive.

[0165] A23. The method according to any one of A to A22, wherein the blocking oligonucleotide comprises a 5' extension template.

[0166] A24. The method according to A23, wherein the 5' extension template is not complementary to the oligonucleotide conjugated to the protein binding reagent.

[0167] A25. The method according to A23 or A24, wherein the length of the 5' extension template is about 10 to about 30 nucleotides.

[0168] A26. The method according to any one of A to A25, wherein the blocking oligonucleotide comprises a 3' flap region.

[0169] A27. The method according to A26, wherein the 3' flap region is not complementary to the oligonucleotide conjugated to the protein binding reagent.

[0170] A28. The method according to A26 or A27, wherein the length of the 3' flap region is about 5 to about 15 nucleotides.

[0171] A29. The method according to any one of A to A28, wherein the blocking oligonucleotide comprises a 5' to 3' structure that comprises (i) a nucleotide sequence comprising an extension template (e.g., a first nucleotide sequence), (ii) a nucleotide sequence complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a second nucleotide sequence), and (iii) a nucleotide sequence not complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a third nucleotide sequence, e.g., a 3' flap region).

[0172] B. The present disclosure further provides a method for imaging a target protein and a target nucleic acid in a sample, the method comprising: (a) binding a protein binding reagent conjugated to an oligonucleotide to the target protein in the sample; (b) hybridizing a blocking oligonucleotide to the oligonucleotide conjugated to the protein binding reagent; (c) amplifying the target nucleic acid in the sample by performing an amplification process to produce an amplicon; (d) imaging the target protein by detecting a first labeled detection probe that hybridizes to the oligonucleotide conjugated to the protein binding reagent; and (e) imaging the target nucleic acid by detecting a second labeled detection probe that hybridizes to the amplicon.

[0173] B1. The method according to B, wherein the amplification process is a rolling circle amplification process.

[0174] B2. The method according to B1, wherein the rolling circle amplification process comprises: (a) contacting the sample with (i) a padlock probe comprising two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to generate a circular DNA template comprising the target nucleic acid; and (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

[0175] B3. The method according to any one of B to B2, wherein the protein binding reagent is an antibody or an antigen-binding fragment thereof.

[0176] B4. The method according to any one of B to B3, wherein the target nucleic acid comprises RNA.

[0177] B5. The method according to any one of B to B4, wherein at least 10 target proteins are imaged in the sample.

[0178] B6. The method according to any one of B to B5, wherein at least 10 target nucleic acids are imaged in the sample.

[0179] B7. The method according to any one of B to B6, wherein the sample is post-fixed before amplifying the target nucleic acid.

[0180] B8. The method according to any one of B to B7, wherein the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid.

[0181] B9. The method according to any one of B to B8, wherein the sample is treated with NHS-acetic acid before amplifying the target nucleic acid.

[0182] B10. The method according to any one of B to B9, wherein the sample is a tissue sample.

[0183] B11. The method according to any one of B to B10, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent is located at the 5' end of the blocking oligonucleotide.

[0184] B12. The method according to any one of B to B11, wherein the oligonucleotide conjugated to the protein binding reagent comprises a barcode sequence, and wherein the blocking oligonucleotide does not bind to the barcode sequence.

[0185] B13. The method according to any one of B to B12, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

[0186] B14. The method according to any one of B to B13, wherein one or more modified nucleotides are located at the 3' end of the blocking oligonucleotide, for example, within the 3' flap region.

[0187] B15. The method according to any one of B to B12, wherein the blocking oligonucleotide comprises at least about 10 modified nucleotides.

[0188] B16. The method according to B15, wherein at least about 10 modified nucleotides are located at the 3' end of the blocking oligonucleotide, for example, within the 3' flap region.

[0189] B17. The method according to any one of B13 to B116, wherein one or more modified nucleotides and / or at least 10 of the modified nucleotides comprise nucleotides having a phosphodiester backbone modification (e.g., phosphorothioate bond).

[0190] B18. The method according to any one of B to B17, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent is about 50 to about 100 nucleotides in length.

[0191] B19. The method according to any one of B to B17, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent is about 10 to about 40 nucleotides in length.

[0192] B20. The method according to any one of B to B17, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent is about 20 to about 30 nucleotides in length.

[0193] B21. The method according to B18, B19 or B20, wherein about 50 to about 100 nucleotides, about 10 to about 40 nucleotides and / or about 20 to about 30 nucleotides are contiguous.

[0194] B22. The method according to any one of B to B21, wherein the blocking oligonucleotide comprises a 5' extension template.

[0195] B23. The method according to B22, wherein the 5' extension template is not complementary to the oligonucleotide conjugated to the protein binding reagent.

[0196] B24. The method according to B22 or B23, wherein the 5' extension template is about 10 to about 30 nucleotides in length.

[0197] B25. The method according to any one of B to B24, wherein the blocking oligonucleotide comprises a 3' flap region.

[0198] B26. The method according to B25, wherein the 3' flap region is not complementary to the oligonucleotide conjugated to the protein binding reagent.

[0199] B27. The method according to B25 or B26, wherein the length of the 3' flap region is from about 5 to about 15 nucleotides.

[0200] B28. The method according to any one of B to B27, wherein the blocking oligonucleotide comprises a 5' to 3' structure that comprises (i) a nucleotide sequence that comprises an extended template (e.g., a first nucleotide sequence), (ii) a nucleotide sequence that is complementary to an oligonucleotide conjugated to a protein binding reagent (e.g., a second nucleotide sequence), and (iii) a nucleotide sequence that is not complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a third nucleotide sequence, e.g., a 3' flap region).

[0201] C. The present disclosure provides a method for imaging a target protein and a target nucleic acid in a sample, the method comprising: (a) providing a sample; (b) contacting the sample with a protein binding reagent that specifically binds to the target protein in the sample, wherein the protein binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide (e.g., an exonuclease blocking oligonucleotide) that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein binding reagent; (d) amplifying the target nucleic acid in the sample by performing an amplification process to produce an amplicon; (e) contacting the sample with a first bridging oligonucleotide that comprises a sequence complementary to the sequence of the oligonucleotide conjugated to the protein binding reagent, and contacting the sample with a first labeled detection probe that comprises a sequence complementary to the sequence of the first bridging oligonucleotide; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second bridging oligonucleotide that comprises a sequence complementary to the sequence of the amplicon, and contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the second bridging oligonucleotide; and (h) imaging the second labeled detection probe to detect the target nucleic acid.

[0202] C1. The method according to C, wherein the amplification process is a rolling circle amplification process.

[0203] C2. The method according to C1, wherein the rolling circle amplification process comprises: (a) contacting the sample with (i) a padlock probe that comprises two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to produce a circular DNA template; and (b) performing a rolling circle amplification process to produce an amplicon from the circular DNA template.

[0204] C3. The method according to any one of C to C2, wherein the protein binding reagent is an antibody or an antigen-binding fragment thereof.

[0205] C4. The method according to any one of C to C3, wherein the target nucleic acid comprises RNA.

[0206] C5. The method according to any one of C to C4, wherein providing the sample comprises one or more of the following: (a) treating the sample with a fixative; (b) dehydrating the sample; and (c) permeabilizing the sample.

[0207] C6. The method according to any one of C to C5, wherein at least 10 target proteins are imaged in the sample.

[0208] C7. The method according to any one of C to C6, wherein at least 10 target nucleic acids are imaged in the sample.

[0209] C8. The method according to any one of C to C7, wherein the sample is post-fixed before amplifying the target nucleic acid.

[0210] C9. The method according to any one of C to C8, wherein the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid.

[0211] C10. The method according to any one of C to C9, wherein the sample is treated with NHS-acetic acid before amplifying the target nucleic acid.

[0212] C11. The method according to any one of C to C10, wherein the sample is a tissue sample.

[0213] C12. The method according to any one of C to C11, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent is located at the 5' end of the blocking oligonucleotide.

[0214] C13. The method according to any one of C to C12, wherein the oligonucleotide conjugated to the protein binding reagent comprises a barcode sequence, and wherein the blocking oligonucleotide does not bind to the barcode sequence.

[0215] C14. The method according to any one of C to C13, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

[0216] C15. The method according to any one of C to C14, wherein one or more modified nucleotides are located at the 3' end of the blocking oligonucleotide, e.g., within the 3' flap region.

[0217] C16. The method according to any one of C to C13, wherein the blocking oligonucleotide comprises at least about 10 modified nucleotides.

[0218] C17. The method according to C16, wherein at least about 10 modified nucleotides are located at the 3' end of the blocking oligonucleotide, e.g., within the 3' flap region.

[0219] C18. The method according to any one of C14 to C17, wherein the one or more modified nucleotides and / or at least 10 of the modified nucleotides comprise nucleotides having a phosphodiester backbone modification (e.g., phosphorothioate bond).

[0220] C19. The method according to any one of C to C18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent has a length of about 50 to about 100 nucleotides.

[0221] C20. The method according to any one of C to C18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent has a length of about 10 to about 40 nucleotides.

[0222] C21. The method according to any one of C to C18, wherein the nucleotide sequence of the blocking oligonucleotide complementary to the oligonucleotide conjugated to the protein binding reagent has a length of about 20 to about 30 nucleotides.

[0223] C22. The method according to C19, C20 or C21, wherein the about 50 to about 100 nucleotides, about 10 to about 40 nucleotides and / or about 20 to about 30 nucleotides are contiguous.

[0224] C23. The method according to any one of C to C22, wherein the blocking oligonucleotide comprises a 5' extension template.

[0225] C24. The method according to C23, wherein the 5' extension template is not complementary to the oligonucleotide conjugated to the protein binding reagent.

[0226] C25. The method according to C23 or C24, wherein the 5' extension template has a length of about 10 to about 30 nucleotides.

[0227] C26. The method according to any one of C to C25, wherein the blocking oligonucleotide comprises a 3' flap region.

[0228] C27. The method according to C26, wherein the 3' flap region is not complementary to the oligonucleotide conjugated to the protein binding reagent.

[0229] C28. The method according to C26 or C27, wherein the 3' flap region has a length of about 5 to about 15 nucleotides.

[0230] C29. The method according to any one of C to C28, wherein the blocking oligonucleotide comprises a 5' to 3' structure that includes (i) a nucleotide sequence containing an extension template (e.g., a first nucleotide sequence), (ii) a nucleotide sequence complementary to an oligonucleotide conjugated to a protein binding reagent (e.g., a second nucleotide sequence), and (iii) a nucleotide sequence not complementary to the oligonucleotide conjugated to the protein binding reagent (e.g., a third nucleotide sequence, e.g., a 3' flap region).

[0231] D. The present disclosure further provides a kit or system for performing the method according to any one of A to C29.

[0232] D1. The kit or system according to D, which includes at least one container containing a blocking oligonucleotide.

[0233] Example

[0234] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided as illustrative of the presently disclosed subject matter and not as a limitation.

[0235] Example 1: Simultaneous Imaging of RNA and Protein in a Sample

[0236] This example describes a method for detecting RNA and protein in a single biological sample, as Figure 1 shown.

[0237] Sample Preparation:

[0238] The method begins with preparing a sample, e.g., a section of the sample, for protein and nucleic acid detection. If the sample is frozen, the sample can be thawed for 5 minutes at -80 °C in a covered chamber. Sample preparation is initiated by fixing the sample in 4% formaldehyde in 1X PBS for 5 minutes to 1 hour. The sample is dehydrated with an ethanol series (i.e., 70%, 85%, and 100%) for 1 minute each. Then the sample is dried at RT in a closed container for 5 minutes or until dry. The sample is mounted into a flow cell and washed 2 times with PBS-Tween (PBST).

[0239] Antibody Staining:

[0240] Subsequently, the sample was stained for proteins by using an antibody specific for the protein and conjugated to an oligonucleotide, where the oligonucleotide includes a barcode sequence. The sample was permeabilized for 20 minutes with PBS+TritonX (0.5%)+1:80 RiboLock. The sample was blocked for 15 minutes to overnight at 4°C or room temperature and stained with one or more Total-Seq antibodies for 1.5 hours to overnight at 4°C or room temperature. The blocking solution further includes an oligonucleotide complementary to a conserved sequence in the oligonucleotide conjugated to the antibody. It has been found that in many tissue types, making RNA accessible requires treatment with hydrochloric acid (HCl) and often protease (as described below), and such treatment makes it difficult to detect RNA and proteins in a single sample by imaging. In particular, the HCl treatment used to permeabilize the sample for RNA detection eliminates antibody binding. Therefore, to maintain antibody attachment during RNA staining, the sample was subsequently washed 2 times with PBST and post-fixed for 5 minutes to 1 hour in 4% PFA or in 4% PFA and 1% glutaraldehyde, and washed 3 times with PBST. This post-fixation treatment retains the antibody while facilitating RNA hybridization, even in HCl-denatured tissues.

[0241] mRNA detection:

[0242] After protein staining, the sample was stained for mRNA by the following protocol. As described above, the antibody-stained sample was permeabilized in 0.1N HCl for 3 minutes. The solution containing 0.1N HCl may also include 1 mg / ml pepsin. HCl permeabilization was performed after antibody staining because it was found that treatment with HCl before antibody staining significantly reduced and modified the antibody staining pattern. It was also found that after the pretreatment steps for protein and RNA staining, RNA staining in the tissue was not ideal. To promote improved staining, the sample was treated with NHS-acetate before RNA hybridization but after HCl permeabilization. In particular, the sample was washed 5 times with PBST and treated with NHS-acetate for 30 minutes.

[0243] Then the sample was blocked for 30 minutes in hybridization blocking buffer and Phi29 blocking oligonucleotide and washed 2 times in PBST. The Phi29 blocking oligonucleotide may have Figure 4 the structure shown. The Phi29 blocking oligonucleotide is used to protect the oligonucleotide conjugated to the antibody from Phi29 3'→5' exonuclease activity, which can rapidly degrade the single-stranded 3' end of the oligonucleotide, as Figure 4as shown in the right figure. The Phi29-blocking oligonucleotide has two features: (1) a long 5' extended template, which is found to block the exonuclease activity of Phi29 on the antibody-oligonucleotide conjugate, and (2) a 3' flap with at least 10 phosphorothioate bonds, which is found to prevent Phi29 from generating DNA barcode duplexes and maintain the hybridization readout of DNA barcodes. The 3' flap of the Phi29-blocking oligonucleotide can have a length of about 10 - 20 nucleotides with at least 10 phosphorothioate bonds. The 5' extended template of the Phi29-blocking oligonucleotide can have a length of about 15 - 25 nucleotides, such as about 22 nucleotides. The length of the nucleotide sequence present in the Phi29-blocking oligonucleotide complementary to the oligonucleotide of the antibody-oligonucleotide conjugate can be about 20 - 30 nucleotides. As Figure 4 shown, the use of the Phi29-blocking oligonucleotide greatly improves the sensitivity of protein detection.

[0244] The sample was then hybridized in 20% formamide hybridization buffer and 10 nM padlock oligonucleotide at 45 °C for 16 - 18 hours. The sample was washed 3 times with PBST. Ligation was carried out overnight with SPLINTR ligase at RT and then the sample was washed 3 times with PBST. Rolling circle amplification (RCA) was carried out overnight at 30 °C. The sample was washed 2 times with PBST and post-fixed in 4% PFA for 15 minutes. During RCA, the polymerase activity of Phi29 polymerase extends the 3' end of the oligonucleotide conjugated to the antibody to generate a complementary sequence to the 5' extended template of the Phi29-blocking oligonucleotide, as Figure 4 shown.

[0245] Cyclic imaging:

[0246] As Figure 2 、 Figure 3 and Figure 5 shown, imaging of target proteins and target nucleic acids is performed by cyclic imaging.

[0247] For protein detection, the protein detection bridge oligonucleotide and the accompanying readout oligonucleotide hybridize with the oligonucleotide conjugated to the antibody, as Figure 1As shown, the protein detection bridge oligonucleotide binds to the barcode sequence on the antibody-oligonucleotide conjugate and five nucleotides of the conserved sequence on the oligonucleotide to maximize stability. The protein detection bridge oligonucleotide has a nucleotide sequence of approximately 28 nucleotides that is complementary to the oligonucleotide of the antibody-oligonucleotide conjugate. The protein detection bridge oligonucleotide includes a repeat sequence of the nucleotide sequence that binds to the fluorescently labeled probe to amplify the detected fluorescent signal. The sample is then washed and imaged. This cycle is repeated until all target proteins are imaged. To remove the probe and the protein detection bridge oligonucleotide prior to the next imaging cycle, heat denaturation is performed. Alternatively, a probe with a disulfide-conjugated dye is used, which allows cleavage of the disulfide to remove the fluorescent signal from the probe.

[0248] For mRNA detection, the mRNA detection bridge oligonucleotide and the accompanying readout oligonucleotide are hybridized to the target mRNA. The mRNA detection bridge oligonucleotide includes a nucleotide sequence complementary to the amplicon generated during RCA (e.g., the sequence present in the padlock oligonucleotide), and also includes a repeat sequence of the nucleotide sequence that binds to the fluorescently labeled probe to amplify the detected fluorescent signal. The mRNA detection bridge oligonucleotide has a nucleotide sequence of approximately 12 nucleotides that is complementary to the amplicon. The sample is then washed and imaged. This cycle is repeated until all target mRNAs are imaged. As described above for protein detection, heat denaturation or a probe with a disulfide-conjugated dye is used to allow cyclic imaging.

[0249] As Figure 3 and Figure 5 shown, the method allows for the simultaneous detection of multiple proteins and mRNAs in a single sample.

[0250] ********

[0251] Although the presently disclosed subject matter has been described in detail and its advantages, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the disclosure. Additionally, the scope of this application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described in the specification. Accordingly, the appended claims are intended to cover such processes, machines, manufactures, compositions of matter, means, methods, or steps within their scope.

[0252] Various patents, patent applications, publications, product descriptions, and protocols are cited in this application, and for all purposes, their disclosures are incorporated herein by reference in their entirety.

Claims

1. A method for imaging a target protein and a target nucleic acid in a sample, the method comprising: (a) providing a sample; (b) contacting the sample with a protein-binding reagent that specifically binds to the target protein in the sample, wherein the protein-binding reagent is conjugated to an oligonucleotide; (c) contacting the sample with a blocking oligonucleotide that comprises a nucleotide sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (d) amplifying the target nucleic acid in the sample by performing an amplification process to generate amplicons; (e) contacting the sample with a first labeled detection probe that comprises a sequence complementary to the sequence of the oligonucleotide conjugated to the protein-binding reagent; (f) imaging the first labeled detection probe to detect the target protein; (g) contacting the sample with a second labeled detection probe that comprises a sequence complementary to the sequence of the amplicon; and (h) imaging the second labeled detection probe to detect the target nucleic acid.

2. The method according to claim 1, wherein the amplification process is a rolling circle amplification process.

3. The method according to claim 2, wherein the rolling circle amplification process comprises: (a) contacting the sample with (i) a padlock probe comprising two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to generate a circular DNA template; and (b) performing a rolling circle amplification process to generate amplicons from the circular DNA template.

4. The method according to any one of claims 1 to 3, wherein the protein-binding reagent is an antibody or an antigen-binding fragment thereof.

5. The method according to any one of claims 1 to 4, wherein the target nucleic acid comprises RNA.

6. The method according to any one of claims 1 to 4, wherein the target nucleic acid is the oligonucleotide conjugated to the protein-binding reagent, the blocking oligonucleotide, or a combination thereof.

7. The method according to any one of claims 1 to 6, wherein the nucleotide sequence complementary to the oligonucleotide conjugated to the protein-binding reagent is located at the 5' end of the blocking oligonucleotide.

8. The method according to any one of claims 1 to 7, wherein the oligonucleotide conjugated to the protein-binding reagent comprises a barcode sequence, and wherein the blocking oligonucleotide does not bind to the barcode sequence.

9. The method according to any one of claims 1 to 8, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

10. The method according to claim 9, wherein the one or more modified nucleotides are located at the 3' end of the blocking oligonucleotide.

11. The method according to claim 9 or 10, wherein the one or more modified nucleotides comprise nucleotides having phosphorothioate bonds.

12. The method according to any one of claims 1 to 12, wherein the 3' end of the blocking oligonucleotide comprises from about 1 to about 10 nucleotides having phosphorothioate bonds.

13. The method according to any one of claims 1 to 12, wherein providing the sample comprises one or more of the following: (a) treating the sample with a fixative; (b) dehydrating the sample; and (c) permeabilizing the sample.

14. The method according to any one of claims 1 to 13, wherein at least 10 target proteins are imaged in the sample.

15. The method according to any one of claims 1 to 14, wherein at least 10 target nucleic acids are imaged in the sample.

16. The method according to any one of claims 1 to 15, wherein the sample is post-fixed before amplifying the target nucleic acid.

17. The method according to any one of claims 1 to 16, wherein the sample is permeabilized with hydrochloric acid before amplifying the target nucleic acid.

18. The method according to any one of claims 1 to 17, wherein the sample is treated with NHS-acetic acid before amplifying the target nucleic acid.

19. The method according to any one of claims 1 to 18, wherein the sample is a tissue sample.

20. A method for imaging target proteins and target nucleic acids in a sample, the method comprising: (a) binding a protein-binding reagent conjugated to an oligonucleotide to a target protein in the sample; (b) hybridizing a blocking oligonucleotide to the oligonucleotide conjugated to the protein-binding reagent; (c) amplifying a target nucleic acid in the sample by performing an amplification process to produce an amplicon; (d) imaging the target protein by detecting a first labeled detection probe that hybridizes to the oligonucleotide conjugated to the protein-binding reagent; and (e) imaging the target nucleic acid by detecting a second labeled detection probe that hybridizes to the amplicon.

21. The method according to claim 20, wherein the amplification process is a rolling circle amplification process.

22. The method according to claim 21, wherein the rolling circle amplification process comprises: (a) contacting the sample with (i) a padlock probe comprising two nucleotide sequences complementary to the target nucleic acid and (ii) a ligase to produce a circular DNA template comprising the target nucleic acid; and (b) performing a rolling circle amplification process to produce an amplicon from the circular DNA template.

23. The method according to any one of claims 20 to 22, wherein the protein-binding reagent is an antibody or an antigen-binding fragment thereof.

24. The method according to any one of claims 20 to 23, wherein the target nucleic acid comprises RNA.

25. The method according to any one of claims 20 to 23, wherein the target nucleic acid is the oligonucleotide conjugated to the protein-binding reagent, the blocking oligonucleotide, or a combination thereof.

26. The method according to any one of claims 20 to 25, wherein the nucleotide sequence complementary to the oligonucleotide conjugated to the protein-binding reagent is located at the 5' end of the blocking oligonucleotide.

27. The method according to any one of claims 20 to 26, wherein the oligonucleotide conjugated to the protein binding reagent comprises a barcode sequence, and wherein the blocking oligonucleotide does not bind to the barcode sequence.

28. The method according to any one of claims 20 to 27, wherein the blocking oligonucleotide comprises one or more modified nucleotides.

29. The method according to claim 28, wherein the one or more modified nucleotides are located at the 3'-end of the blocking oligonucleotide.

30. The method according to claim 28 or 29, wherein the one or more modified nucleotides comprise nucleotides having phosphorothioate bonds.

31. The method according to any one of claims 20 to 30, wherein at least 10 target proteins are imaged in the sample.

32. The method according to any one of claims 20 to 31, wherein at least 10 target nucleic acids are imaged in the sample.

33. The method according to any one of claims 20 to 32, wherein the sample is post-fixed before amplification of the target nucleic acid.

34. The method according to any one of claims 20 to 33, wherein the sample is permeabilized with hydrochloric acid before amplification of the target nucleic acid.

35. The method according to any one of claims 20 to 34, wherein the sample is treated with NHS-acetic acid before amplification of the target nucleic acid.

36. The method according to any one of claims 20 to 35, wherein the sample is a tissue sample.

37. A kit for performing the method according to any one of claims 1 to 36.

38. The kit according to claim 37, comprising at least one container containing the blocking oligonucleotide.

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