Method and system for removing histological stains from a sample

Abstract

The present disclosure relates to a method and a composition for removing histological staining from a histologically stained sample. This method can remove histological staining from a histologically stained sample by including contacting the sample with an acidifying agent and contacting the sample with a reducing agent.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 347,150, filed May 31, 2022, and U.S. Provisional Patent Application No. 63 / 434,578, filed December 22, 2022, the entire contents of each of which are hereby incorporated by reference for all purposes.

[0002] Field The present disclosure relates to methods and compositions for removing histological stains from biological samples. The methods and compositions can be used, for example, to remove hematoxylin from tissue samples.

Background Art

[0003] Hematoxylin and eosin (H&E) staining is well - established in the field of histopathology. H&E staining is used for the evaluation of cell morphology and is the primary tool used by pathologists to diagnose cancer. To obtain additional information about the pathology, other analytical methods such as immunohistochemistry (IHC) and in - situ hybridization are commonly performed to search for protein or nucleic acid targets. This analysis is often performed on adjacent thin sections cut from the same tissue sample. However, such adjacent sections differ from each other at the single - cell level, and tissue samples can be in short supply (e.g., tissue from a needle biopsy), and as a result, techniques for using a single tissue section for multiple analyses are of interest.

[0004] One of the main problems associated with using H&E-stained samples for subsequent analysis is interference from H&E staining when detecting subsequent labels used in methods such as IHC and in situ hybridization, e.g., chromogenic or fluorescent labels. In some cases, attempts have been made to perform fluorescent IHC using near-infrared dyes with emission far from H&E fluorescence. However, the spectral space available for such dyes is very limited. Most commercially available fluorescent probes and labeled antibodies are labeled with fluorophores that have emission overlapping with H&E emission.

[0005] It has been reported that subsequent IHC staining can be performed on H&E-decolorized tissue sections by a method of removing H&E staining. However, in multiplex chromogenic IHC and fluorescent IHC, such attempts have not been successful. This is because, for example, hematoxylin and eosin cannot be completely removed using existing methods. Even a small amount of residual hematoxylin staining can be demonstrated to extensively interfere with the detection of fluorescent signals above the background. In some examples, workflows for removing hematoxylin using strong acids have been proposed. However, the time required for removal can take from 1 hour to several hours, and such long-term treatment with strong acids can degrade the sample and make it unsuitable for subsequent analysis using other methods.

[0006] Therefore, there is still a need for a method to efficiently remove bound hematoxylin from biological samples, e.g., for subsequent analysis such as IHC or in situ hybridization, and other chromogenic and fluorescence-based methods for detecting biomolecules such as nucleic acids and proteins, so that the same sample can be used. SUMMARY OF THE INVENTION

[0007] The present disclosure relates to methods and compositions for removing histological stains (e.g., hematoxylin) from stained biological samples such as cell- or tissue-containing samples. In certain embodiments, removal of the hematoxylin stain is achieved by treatment with a strong acid followed by treatment with a reducing agent. In some embodiments, this process is achieved in less than 1 hour.

[0008] The present disclosure also provides a method for detecting multiple targets in a sample. The method can optionally include detecting the hematoxylin stain of the sample, removing the hematoxylin stain from the stained sample, and detecting additional features or targets in the sample. The additional features can include any morphological features such as connective tissue, fat, amyloid, myelin, etc. that can be detected using an analytical method. The additional targets can include any protein, nucleic acid, or any other molecular target that can be detected using an analytical method.

[0009] Accordingly, the following embodiments are provided in accordance with this description.

[0010] Embodiment 1 is a method for removing histological stains from a histologically stained sample, comprising: (a) contacting the sample with an acidifying agent having a pH from 1.0 to 4.0; and (b) contacting the sample with a reducing agent having a pH from 8.0 to 10.0, thereby removing the histological stain from the histologically stained sample.

[0011] Embodiment 1A is a method for removing histological stains from a histologically stained sample, comprising: (a) contacting the sample with an acidifying agent having a pH from 1.0 to 4.0; (b) removing the acidifying agent; and (c) contacting the sample with a reducing agent having a pH from 8.0 to 10.0, thereby removing the histological stain from the histologically stained sample.

[0012] Embodiment 2 is a method for preparing a histologically stained sample for a subsequent analysis method for detecting at least one target, comprising: (a) contacting the sample with an acidic agent having a pH from 1.0 to 4.0; (b) contacting the sample with a reducing agent having a pH from 8.0 to 10.0; (c) contacting the sample with a staining agent to stain at least one target in the sample; and (d) optionally, detecting the at least one target in the sample.

[0013] Embodiment 2A is a method for preparing a histologically stained sample for a subsequent analysis method for detecting at least one target, comprising: (a) contacting the sample with an acidic agent having a pH from 1.0 to 4.0; (b) removing the acidic agent; (c) contacting the sample with a reducing agent having a pH from 8.0 to 10.0; (d) removing the reducing agent; (e) contacting the sample with a staining agent to specifically stain at least one target in the sample; and (f) optionally, detecting the at least one target in the sample.

[0014] Embodiment 3 is the method according to Embodiment 1 or 2, wherein the acidic agent is removed from the sample. In some embodiments, the acidic agent is removed from the sample using a rinsing agent.

[0015] Embodiment 4 is the method according to Embodiment 2, wherein the reducing agent is removed from the sample. In some embodiments, the reducing agent is removed from the sample using a rinsing agent.

[0016] Embodiment 5 is the method according to any one of the preceding embodiments, wherein the histological staining comprises hematoxylin.

[0017] Embodiment 6 is the method according to Embodiment 5, wherein the histological staining comprises hematoxylin and eosin.

[0018] Embodiment 7 is the method according to Embodiment 6, further including detecting hematoxylin and eosin staining before step (a).

[0019] Embodiment 8 is the method according to any one of the preceding embodiments, wherein the pH of the acidifying agent is from 1.0 to 2.0.

[0020] Embodiment 9 is the method according to any one of the preceding embodiments, wherein the acidifying agent includes hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydroiodic acid, sulfamic acid, perchloric acid, or a combination thereof.

[0021] Embodiment 10 is the method according to Embodiment 9, wherein the acidifying agent includes hydrochloric acid (HCl).

[0022] Embodiment 11 is the method according to any one of the preceding embodiments, wherein the acidifying agent includes a solvent.

[0023] Embodiment 12 is the method according to Embodiment 11, wherein the solvent of the acidifying agent includes a solvent selected from water, ethylene glycol, polyethylene glycol, propylene glycol, ethanol, methanol, or a combination thereof.

[0024] Embodiment 13 is the method according to Embodiment 12, wherein the solvent of the acidifying agent includes water.

[0025] Embodiment 14 is the method according to Embodiment 12, wherein the solvent of the acidifying agent includes ethylene glycol.

[0026] Embodiment 15 is the method according to Embodiment 12, wherein the acidifying agent includes 1% HCl in 70% ethanol.

[0027] Embodiment 16 is the method according to any one of the preceding embodiments, wherein the acidifying agent includes the acid at a molar concentration of 0.01 M to 0.5 M.

[0028] Embodiment 17 is the method according to Embodiment 16, wherein the acidifying agent contains 0.12 M hydrochloric acid.

[0029] Embodiment 18 is the method according to any one of the preceding embodiments, wherein the pH of the reducing agent is from 8.0 to 9.0.

[0030] Embodiment 19 is the method according to any one of the preceding embodiments, wherein the reducing agent contains a reducing agent selected from sodium borohydride, sodium cyanoborohydride, potassium bromate, sodium sulfite, sodium dithionite, sodium thiosulfate, sodium bisulfite, sodium triethylborohydride, and sodium triacetoxyborohydride, or a combination thereof.

[0031] Embodiment 20 is the method according to any one of the preceding embodiments, wherein the reducing agent contains a solvent.

[0032] Embodiment 21 is the method according to Embodiment 20, wherein the solvent contains a solvent selected from water, alcohol, ethylene glycol, and propylene glycol, or a combination thereof.

[0033] Embodiment 22 is the method according to Embodiment 21, wherein the solvent of the reducing agent contains water or alcohol, and optionally, the alcohol is selected from ethanol, methanol, isopropanol, or a combination thereof. In a further embodiment, the solvent of the reducing agent contains ethanol.

[0034] Embodiment 23 is the method according to Embodiment 21, wherein the solvent of the reducing agent contains ethylene glycol.

[0035] Embodiment 24 is the method according to Embodiment 21, wherein the reducing agent contains 1% sodium borohydride. In some embodiments, the reducing agent contains 1% sodium borohydride in water or ethanol. In a further embodiment, the reducing agent contains 1% sodium borohydride in water.

[0036] Embodiment 25 is the method according to Embodiment 19, wherein the reducing agent contains sodium borohydride with a molar concentration ranging from 0.05 M to 0.5 M.

[0037] Embodiment 26 is the method according to Embodiment 25, wherein the reducing agent contains 0.26 M of sodium borohydride.

[0038] Embodiment 27 is the method according to any one of the preceding embodiments, wherein the step of contacting the sample with the acidifying agent is performed for 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute or less.

[0039] Embodiment 28 is the method according to Embodiment 27, wherein the step of contacting the sample with the acidifying agent includes contacting the sample with the acidifying agent for a period ranging from 2 minutes to 20 minutes, from 2 minutes to 15 minutes, from 2 minutes to 10 minutes, from 2 minutes to 5 minutes, from 2 minutes to 4 minutes, from 2 minutes to 3 minutes, or from 1 minute to 2 minutes.

[0040] Embodiment 29 is the method according to Embodiment 28, wherein the step of contacting the sample with the reducing agent is performed for 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, or 2 minutes or less.

[0041] Embodiment 30 is the method according to any one of the preceding embodiments, wherein the step of contacting the sample with the reducing agent includes contacting the sample with the reducing agent for a period ranging from 2 minutes to 20 minutes, from 2 minutes to 15 minutes, from 2 minutes to 10 minutes, from 2 minutes to 8 minutes, from 2 minutes to 7 minutes, or from 2 minutes to 5 minutes.

[0042] Embodiment 31 is the method according to any one of the preceding embodiments, wherein the step of contacting the sample with the acidifying agent or the reducing agent is performed for 40 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, or 5 minutes or less.

[0043] Embodiment 32 is the method according to any one of the preceding embodiments, wherein during the step of contacting the sample with the acidifying agent, the temperature of the sample is maintained between about 0°C and 10°C, between about 20°C and 30°C, between about 35°C and about 100°C, between about 40°C and about 90°C, between about 45°C and about 80°C, or between about 50°C and about 70°C.

[0044] Embodiment 33 is the method according to Embodiment 32, wherein during the step of contacting the sample with the acidifying agent, the temperature of the sample is maintained between about 20°C and 30°C.

[0045] Embodiment 34 is the method according to any one of the preceding embodiments, further comprising applying heat to the sample so that the sample and the acidifying agent are maintained at a predetermined temperature during contact during the step of contacting the sample with the acidifying agent.

[0046] Embodiment 35 is the method according to any one of the preceding embodiments, wherein during the step of contacting the sample with the reducing agent, the temperature of the sample is maintained between about 0°C and 10°C, between about 20°C and 30°C, between about 35°C and about 100°C, between about 40°C and about 90°C, between about 45°C and about 80°C, or between about 50°C and about 70°C.

[0047] Embodiment 36 is the method according to Embodiment 35, wherein during the step of contacting the sample with the reducing agent, the temperature of the sample is maintained between about 20°C and 30°C.

[0048] Embodiment 37 is the method according to any one of the preceding embodiments, further comprising applying heat to the sample so that the sample and the reducing agent are maintained at a predetermined temperature during contact during the step of contacting the sample with the reducing agent.

[0049] Embodiment 38 is the method according to any one of Embodiments 2 to 37, wherein the staining agent comprises using an immunohistochemical reagent, an in situ hybridization reagent, or a combination thereof.

[0050] Embodiment 39 is: (1) contacting the tissue sample that has been tested for the presence of at least one target with at least one corresponding target-specific binding partner, wherein each target-specific binding partner with a different specificity is linked to a different nucleic acid strand, said contacting; (2) contacting the tissue sample with a labeled imager strand that is complementary to the nucleic acid strand linked to the target-specific binding partner; (3) imaging the sample to detect the labeled bound imager strand; (4) optionally, removing the signal from the labeled bound imager strand; (5) optionally, repeating at least a part of steps (2) to (4) at least once using a labeled imager strand having a unique composition as compared with at least one other labeled imager strand to detect the location of the at least one target in the sample, and is the method according to any one of the preceding claims.

[0051] Embodiment 40 is the method according to any one of the preceding embodiments, wherein the sample contains cells.

[0052] Embodiment 41 is the method according to any one of the preceding embodiments, wherein the sample contains tissue.

[0053] Embodiment 42 is the method according to Embodiment 41, wherein the sample is a tissue section.

[0054] Embodiment 43 is the method according to Embodiment 42, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) tissue section.

[0055] Embodiment 44 is the method according to any one of the preceding embodiments, further comprising deparaffinizing and rehydrating the sample before step (a).

[0056] Embodiment 45 is a kit for removing histological stains from tissue samples, the kit comprising: (a) an acid having a pH from 1.0 to 4.0; (b) a reducing agent having a pH from 8.0 to 10.0; (c) optionally, a rinsing agent; and (d) optionally, a staining reagent.

[0057] Embodiment 46 is the kit according to Embodiment 45, wherein the agents (a), (b), and optionally (c) and (d) are separately contained in respective containers.

[0058] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description serve to explain the principles disclosed herein.

[0059] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0060] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0061] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description serve to explain the principles disclosed herein.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0063] I. Exemplary Methods In some embodiments, a method for removing histological stains from histologically stained samples is provided.

[0064] In certain embodiments, the removal of histological staining is achieved by: (a) contacting the sample with an acidic agent; (b) removing the acidic agent; and (c) contacting the sample with a reducing agent. In some embodiments, the acidic agent has a pH from 1.0 to 4.0. In some embodiments, the reducing agent has a pH from 8.0 to 10.0. In some embodiments, the step of contacting the sample with the acidic agent is performed before contacting the sample with the reducing agent. In other embodiments, the step of contacting the sample with the reducing agent is performed before contacting the sample with the acidic agent.

[0065] In some embodiments, the step of contacting the sample of the method disclosed herein with the acidic agent is performed for about 20 minutes, 15, 10, 5, 4, 3, 2, or 1 minute or less. In some embodiments, the method disclosed herein includes contacting the sample with the acidic agent for a period of about 2 minutes to about 20 minutes, about 2 minutes to about 15 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 5 minutes, about 2 minutes to about 4 minutes, about 2 minutes to about 3 minutes, or about 1 minute to about 2 minutes. In some embodiments, step (a) of contacting with the acidic agent is performed for about 2 minutes.

[0066] In some embodiments, the step of contacting the sample with the reducing agent is performed for about 20 minutes, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 minutes or less. In some embodiments, the step of contacting the sample with the reducing agent includes contacting the sample with the reducing agent for a period of about 2 minutes to about 20 minutes, about 2 minutes to about 15 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 8 minutes, about 2 minutes to about 7 minutes, or about 2 minutes to about 5 minutes. In some embodiments, the step of contacting the sample with the reducing agent is performed for about 5 minutes.

[0067] In some embodiments, the treatment with both an acidifying agent and a reducing agent by the methods disclosed herein is carried out for about 40 minutes or less, about 30 minutes or less, about 25 minutes or less, about 10 minutes or less, or about 5 minutes or less. In existing methods of removing hematoxylin using strong acids, it can take at least 1 hour up to 1 or 2 days, and such long-term treatment of samples with acids can degrade the samples and make them unsuitable for subsequent analysis using, for example, fluorescence detection. According to the methods and compositions of the present disclosure, the time for the de-staining process can be significantly reduced.

[0068] In some embodiments, during the step of contacting the sample with the acidifying agent, the temperature of the sample is maintained between about 0°C and 10°C, between about 20°C and 30°C, between about 35°C and about 100°C, between about 40°C and about 90°C, between about 45°C and about 80°C, or between about 50°C and about 70°C. In some embodiments, during the step of contacting the sample with the acidifying agent, the temperature of the sample is maintained between 20°C and 30°C. In some embodiments, the methods disclosed herein further include applying heat to the sample such that the sample and the acidifying agent are maintained at a predetermined temperature during contact between the sample and the acidifying agent. The predetermined temperature may be between about 0°C and 10°C, between about 20°C and 30°C, between about 35°C and about 100°C, between about 40°C and about 90°C, between about 45°C and about 80°C, or between about 50°C and about 70°C.

[0069] In some embodiments, during the step of contacting the sample with the reducing agent, the temperature of the sample is maintained between about 0°C and 10°C, between about 20°C and 30°C, between about 35°C and about 100°C, between about 40°C and about 90°C, between about 45°C and about 80°C, or between about 50°C and about 70°C. In some embodiments, during the step of contacting the sample with the reducing agent, the temperature of the sample is maintained between 20°C and 30°C. In some embodiments, the method disclosed herein further includes applying heat to the sample such that the sample and the reducing agent are maintained at a predetermined temperature during contact. The predetermined temperature may be between about 0°C and 10°C, between about 20°C and 30°C, between about 35°C and about 100°C, between about 40°C and about 90°C, between about 45°C and about 80°C, or between about 50°C and about 70°C.

[0070] In some embodiments, a heating slide platform is used to apply heat to the sample.

[0071] In some embodiments, the sample includes cells. In some embodiments, the sample is a tissue-containing sample or a tissue section. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) tissue section. In some embodiments, the sample has been deparaffinized and rehydrated prior to step (a). In some embodiments, the method further includes deparaffinizing and rehydrating the sample prior to step (a).

[0072] A. Destaining In some embodiments, the histological stain includes hematoxylin.

[0073] As used herein, "H&E" means hematoxylin and eosin. H&E staining involves the use of hematoxylin (the active form of this dye is called hematein, but generally it is referred to as hematoxylin) and eosin. Without wishing to be bound by any theory or mechanism of the present disclosure, during this process hematoxylin is oxidized to hematein, and then hematein binds to aluminum (Al3+ )), iron (Fe 3+ ) and chromium (Cr 3+ ) is considered to bind to metal ions. As a result, the cell nucleus (and some other cell components such as keratohyalin granules) is stained blue. Therefore, the mechanism of nuclear staining by hematoxylin is that the dye-metal complex binds to arginine-rich basic nuclear proteins such as histones and deoxyribonucleic acid (DNA). After nuclear staining, counterstaining follows with an aqueous or alcoholic solution of eosin, which colors other acidophilic structures in various shades of red, pink, and orange. Acidophilic structures are generally composed of intracellular or extracellular proteins. Other methods involving the use of hematoxylin include Masson trichrome, which is used to stain connective tissue.

[0074] B. Acid Treatment The method according to the present disclosure includes treatment with an acidifying agent. Without wishing to be bound by any theory or mechanism, acid treatment is believed to be effective in interfering with the binding between oxidized hematoxylin and the mordant (e.g., Al 3+ , Fe 3+ , or Cr 3+ ) bound to histones and deoxyribonucleic acid (DNA) in the sample.

[0075] In some embodiments, the acidifying agent has a pH from 1.0 to 4.0. In some embodiments, the acidifying agent has a pH of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0.

[0076] In some embodiments, the acidifying agent has a pH from 1.0 to 2.0. In some embodiments, the acidifying agent has a pH from 1.0 to 1.9, from 1.0 to 1.8, from 1.0 to 1.7, from 1.0 to 1.6, from 1.0 to 1.5, from 1.0 to 1.4, from 1.0 to 1.3, from 1.0 to 1.2, or from 1.0 to 1.1. In some embodiments, the acidifying agent has a pH from 1.1 to 2.0, from 1.2 to 2.0, from 1.3 to 2.0, from 1.4 to 2.0, from 1.5 to 2.0, from 1.6 to 2.0, from 1.7 to 2.0, from 1.8 to 2.0, or from 1.9 to 2.0. In some embodiments, the acidifying agent has a pH from 1.1 to 1.9, from 1.2 to 1.8, from 1.3 to 1.7, or from 1.4 to 1.6.

[0077] In some embodiments, the acidifying agent has a pH from 2.0 to 4.0. In some embodiments, the acidifying agent has a pH from 2.0 to 3.6. In some embodiments, the acidifying agent has a pH from 2.0 to 3.5. In some embodiments, the acidifying agent has a pH from 2.0 to 3.4. In some embodiments, the acidifying agent has a pH from 2.0 to 3.3. In some embodiments, the acidifying agent has a pH from 2.0 to 3.2. In some embodiments, the acidifying agent has a pH from 2.0 to 3.1. In some embodiments, the acidifying agent has a pH from 2.0 to 3.0. In some embodiments, the acidifying agent has a pH from 2.0 to 2.9. In some embodiments, the acidifying agent has a pH from 2.0 to 2.8. In some embodiments, the acidifying agent has a pH from 2.0 to 2.7. In some embodiments, the acidifying agent has a pH from 2.0 to 2.6. In some embodiments, the acidifying agent has a pH from 2.0 to 2.5.

[0078] In some embodiments, the acidic agent has a pH from 2.5 to 3.3. In some embodiments, the acidic agent has a pH from 2.5 to 3.6. In some embodiments, the acidic agent has a pH from 2.5 to 3.5. In some embodiments, the acidic agent has a pH from 2.5 to 3.4. In some embodiments, the acidic agent has a pH from 2.5 to 3.3. In some embodiments, the acidic agent has a pH from 2.5 to 3.2. In some embodiments, the acidic agent has a pH from 2.5 to 3.1. In some embodiments, the acidic agent has a pH from 2.5 to 3.0. In some embodiments, the acidic agent has a pH from 2.5 to 2.9. In some embodiments, the acidic agent has a pH from 2.5 to 2.8. In some embodiments, the acidic agent has a pH from 2.5 to 2.7. In some embodiments, the acidic agent has a pH from 2.5 to 2.6.

[0079] In some embodiments, the acidic agent has a pH from 2.8 to 3.6. In some embodiments, the acidic agent has a pH from 2.8 to 3.5. In some embodiments, the acidic agent has a pH from 2.8 to 3.4. In some embodiments, the acidic agent has a pH from 2.8 to 3.3. In some embodiments, the acidic agent has a pH from 2.8 to 3.2. In some embodiments, the acidic agent has a pH from 2.8 to 3.1. In some embodiments, the acidic agent has a pH from 2.8 to 3.0. In some embodiments, the acidic agent has a pH from 2.8 to 2.9.

[0080] In some embodiments, the acidic agent comprises from about 0.5% to about 2% (v / v) HCl, from about 1% to about 2% (v / v) HCl, or from about 1% to about 1.5% (v / v) HCl. In another embodiment, the acidic agent comprises about 70% (v / v) ethanol.

[0081] In some embodiments, the molar concentration of the acid is from 0.01 M to 0.5 M. In some embodiments, the molar concentration of the acid is from about 0.01 M to 0.4 M, from 0.05 M to 0.4 M, from 0.1 M to 0.4 M, from 0.2 M to 0.4 M, or from 0.2 M to 0.3 M. In some embodiments, the molar concentration of the acid is from about 0.01 M to 0.3 M, from 0.05 M to 0.3 M, from 0.1 M to 0.3 M, or from 0.2 M to 0.3 M. In some embodiments, the molar concentration of the acid is from about 0.01 M to 0.2 M, from 0.05 M to 0.2 M, from 0.1 M to 0.2 M, or from 0.15 M to 0.2 M. In some embodiments, the molar concentration of the acid is from about 0.01 M to 0.2 M, from 0.05 M to 0.2 M, from 0.1 M to 0.2 M, or from 0.15 M to 0.2 M. In some embodiments, the molar concentration of the acid is about 0.01, 0.05, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.50 M. For example, in some embodiments, the acid comprises 0.12 M hydrochloric acid.

[0082] In some embodiments, the acidifying agent comprises an acidifying agent selected from hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydroiodic acid, sulfamic acid, perchloric acid, or combinations thereof. The acid can be any strong acid understood and known in the art. A strong acid is any acid that completely dissociates or ionizes in an aqueous solution.

[0083] In some embodiments, the acidifying agent comprises hydrochloric acid.

[0084] In some embodiments, the acidic agent includes a solvent. In some embodiments, the solvent includes water, ethylene glycol, polyethylene glycol, propylene glycol, ethanol, methanol, or a combination thereof. In some embodiments, the solvent in the acidic agent includes water. In some embodiments, the solvent of the acidic agent includes an alcohol (e.g., ethanol). In some embodiments, the solvent includes ethylene glycol.

[0085] In some embodiments, the acidic agent includes about 1% HCl in about 70% ethanol.

[0086] C. Reducing Agent The method according to the present disclosure uses a reduction reaction. In some embodiments, the method includes contacting the sample with a reducing agent.

[0087] In some embodiments, the reducing agent has a pH from 8.0 to 10.0. It should be noted that the pH of the reducing agent used herein refers to the pH of the solution before being used for the sample. In some embodiments, the reducing agent has a pH from 8.0 to 9.5. In some embodiments, the reducing agent has a pH from 8.0 to 9.2. In some embodiments, the reducing agent has a pH from 8.0 to 9.0. In some embodiments, the reducing agent has a pH from 8.0 to 8.9. In some embodiments, the reducing agent has a pH from 8.0 to 8.8. In some embodiments, the reducing agent has a pH from 8.0 to 8.7. In some embodiments, the reducing agent has a pH from 8.0 to 8.6. In some embodiments, the reducing agent has a pH from 8.0 to 8.5. In some embodiments, the reducing agent has a pH from 8.0 to 8.4. In some embodiments, the reducing agent has a pH from 8.0 to 8.3. In some embodiments, the reducing agent has a pH from 8.0 to 8.2. In some embodiments, the reducing agent has a pH from 8.0 to 8.1.

[0088] In some embodiments, the reducing agent has a pH from 8.1 to 9.0. In some embodiments, the reducing agent has a pH from 8.2 to 9.0. In some embodiments, the reducing agent has a pH from 8.3 to 9.0. In some embodiments, the reducing agent has a pH from 8.4 to 9.0. In some embodiments, the reducing agent has a pH from 8.5 to 9.0. In some embodiments, the reducing agent has a pH from 8.6 to 9.0. In some embodiments, the reducing agent has a pH from 8.7 to 9.0. In some embodiments, the reducing agent has a pH from 8.8 to 9.0. In some embodiments, the reducing agent has a pH from 8.9 to 9.0.

[0089] In some embodiments, the reducing agent has a pH from 8.1 to 8.9. In some embodiments, the reducing agent has a pH from 8.2 to 8.8. In some embodiments, the reducing agent has a pH from 8.3 to 8.7. In some embodiments, the reducing agent has a pH from 8.4 to 8.6.

[0090] In some embodiments, the reducing agent has a pH of about 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0.

[0091] In some embodiments, the reducing agent comprises a reducing agent selected from sodium borohydride, sodium cyanoborohydride, potassium bromate, sodium sulfite, sodium dithionite, sodium thiosulfate, sodium bisulfite, sodium triethylborohydride, and sodium triacetoxyborohydride, or combinations thereof.

[0092] In some embodiments, the reducing agent comprises a solvent. In some embodiments, the solvent comprises water, alcohol, ethylene glycol, propylene glycol, polyethylene glycol, or combinations thereof. In some embodiments, the solvent of the reducing agent comprises water. In some embodiments, the solvent of the reducing agent comprises an alcohol such as one or more of ethanol, isopropanol, and / or methanol. In some embodiments, the solvent of the reducing agent comprises ethylene glycol.

[0093] In some embodiments, the reducing agent comprises about 1% sodium borohydride in ethanol, for example, about 50% ethanol, about 60% ethanol, about 70% ethanol, about 80% ethanol, about 90% ethanol, and about 100% ethanol.

[0094] In some embodiments, the reducing agent comprises about 1% sodium borohydride in water. In some embodiments, the reducing agent comprises about 1% sodium borohydride in ethanol.

[0095] In some embodiments, the reducing agent comprises about 1% sodium borohydride in a carbonate-bicarbonate buffer having a pH of about 9.2.

[0096] In some embodiments, the molar concentration of the reducing agent is from 0.1 M to 0.5 M. In some embodiments, the molar concentration of the reducing agent is from 0.1 M to 0.5 M, from 0.2 M to 0.5 M, from 0.3 M to 0.5 M, or from 0.4 M to 0.5 M. In some embodiments, the molar concentration of the reducing agent is from 0.1 M to 0.4 M, from 0.2 M to 0.4 M, or from 0.3 M to 0.4 M. In some embodiments, the molar concentration of the reducing agent is from 0.1 M to 0.3 M, from 0.15 M to 0.3 M, or from 0.2 M to 0.3 M. In some embodiments, the molar concentration of the reducing agent is about 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.50 M. For example, in some embodiments, the reducing agent comprises about 0.26 M sodium borohydride.

[0097] In some embodiments, the reducing agent comprises from 0.05 M to 0.2 M sodium borohydride (e.g., 0.13 M sodium borohydride). In some embodiments, the reducing agent comprises from 0.1 M to 0.5 M sodium cyanoborohydride (e.g., 0.16 M sodium cyanoborohydride).

[0098] D. Removal In some embodiments, a removal step may be performed.

[0099] As used herein, the term "removing" (or "removal" or "removed") with respect to removing histological stain from a sample means achieving a reduction in the signal relative to background that reduces the amount of histological stain in the sample and enables subsequent analysis of the sample using the same or different analytical methods. Thus, in some embodiments, after the methods disclosed herein are performed, there may be some residual histological stain remaining, but the signal generated by any such residual histological stain can be reduced to a level that does not impair subsequent analysis.

[0100] As used with respect to removing an acidifying agent or a reducing agent from a sample, the term "removing" (or "removal" or "removed") means removing the activity of the acidifying agent or reducing agent in the sample, for example, by physically depleting the agent from the sample, by using a material that renders the agent non-functional (e.g., a neutralizing or decomposing material that renders the agent non-functional), or by using conditions that render the agent non-functional (e.g., a temperature that renders the agent non-functional, photoactivating light, etc.). Thus, removal of an acidifying agent can be effected by addition of a reducing agent.

[0101] In some embodiments, the acidifying agent and / or reducing agent is removed by mechanical means, e.g., by decanting, aspiration, positive pressure, etc. In some embodiments, the acidifying agent and / or reducing agent is removed by neutralization. In some embodiments, the acidifying agent and / or reducing agent is removed by rinsing with a rinsing agent, which is optionally removed thereafter. The rinsing agent may comprise, for example, water (which may be deionized water, a buffer, an alcohol mixture). In some embodiments, the acidifying agent and / or reducing agent remains on the sample, but its functionality is removed, e.g., by dilution, neutralization, decomposition, etc.

[0102] If the method uses a rinsing agent after the acidifying agent and a rinsing agent after the reducing agent, the two rinsing agents may be the same or different.

[0103] E. Embodiments including further analysis As used herein, the term "histologically stained sample" means a biological sample stained with a histological dye. The histologically stained sample may be an archived or stored sample (including, for example, a period ranging from minutes, hours, weeks, months, years, or even decades), or a sample specially prepared for sequential staining using different analytical methods. Using the methods described herein, various histological stains can be removed. In one embodiment, the histological stain is H&E. In some embodiments, the methods disclosed herein can be used in a process that removes H&E staining from a sample and subjects the sample to subsequent analytical methods. Further, the same detection channels used for imaging of H&E staining are made available for detection using another analytical method. In some embodiments, the methods disclosed herein can be used for various analytical methods, such as immunohistochemistry, in situ hybridization, other protein and / or nucleic acid detection methods, or detection of cell characteristics or molecular targets using histological staining.

[0104] Examples of histological staining include hematoxylin and eosin (H&E) staining, Giemsa staining, Gram staining, periodic acid Schiff reaction staining, Papanicolaou staining, Masson trichrome staining, Congo red staining, mucicarmine staining, and silver staining. These can be carried out using known methods (for example, Thompson, Samuel W. Selected Histochemical and Histopathological Methods, Springfield, IL, 1966; Sheehan, D.C. and Hrapchak, B.B.: Theory and Practice of Histotechnology, 2nd Edition; Battelle Memorial Institute, Columbus, OH, 1987; Alturkistani HA, Tashkandi FM, Mohammedsaleh ZM. Histological Stains: A Literature Review and Case Study. Glob J Health Sci. 2015 Jun 25;8(3):72-9. Brown RC, Hopps HC. Staining of bacteria in tissue sections: a reliable gram stain method. Am J Clin Pathol. 1973 Aug;60(2):234-40; Dolan M. The role of the Giemsa stain in cytogenetics. Biotech Histochem. 2011 Apr;86(2):94-7; Al Drees A, Salah Khalil M, Soliman M. Histological and Immunohistochemical Basis of the Effect of Aminoguanidine on Renal Changes Associated with Hemorrhagic Shock in a Rat Model. Acta Histochem Cytochem. 2017 Feb 28;50(1):11-19.).Multiplex immunohistochemical staining can be performed using a variety of methods, including commercially available FlexVUE, FixVUE, and U-VUE kits available from Ultivue, Inc., as well as other methods such as those described in "Overview of multiplex immunohistochemistry / immunofluorescence techniques in the era of cancer immunotherapy" (Cancer Commun (Lond). 2020 Apr;40(4):135-153). By detecting targets in the same sample, additional spatial information regarding the targets in the sample can be provided. The methods disclosed herein can also be used for analytical applications where limited amounts of sample may be available for analysis and the same sample may need to be processed for multiple analyses. Thus, the methods disclosed herein can also facilitate multiple analyses of tissue sections.

[0105] The method can further facilitate analysis based on detection methods where the number of targets that can be detected simultaneously may be limited due to limitations of divisible signals.

[0106] Accordingly, in some embodiments, a method of preparing a histologically stained sample for staining to detect at least one target is provided. In certain embodiments, the method can include (a) contacting the sample with an acidifying agent, (b) contacting the sample with a reducing agent, and (c) staining the sample using a subsequent analytical method.

[0107] In some embodiments, the method further includes detecting hematoxylin and eosin (H&E) staining prior to step (a).

[0108] In some embodiments, the subsequent analysis method step includes contacting the sample with a stain to stain at least one target in the sample. In some embodiments, the methods disclosed herein further include detecting at least one target in the sample. In some embodiments, the sample can then be further subjected to successive staining and detection of the target by multiple rounds of staining and signal removal. Thus, the methods disclosed herein can be used to employ another analysis method, such as immunohistochemistry (IHC), in situ hybridization, or spatial transcriptome analysis, after the slide has been subjected to H&E staining.

[0109] In some embodiments, the subsequent analysis is a staining method.

[0110] In some embodiments, the subsequent analysis method is a histological staining method. In some embodiments, the subsequent analysis method is an immunohistochemical staining method. In some embodiments, the subsequent analysis method is a nucleic acid detection method. In some embodiments, the subsequent analysis method is a protein detection method. In some embodiments, the subsequent analysis method is a spatial transcriptome analysis method. Thus, the subsequent analysis method can target any molecule, including, for example, one or more nucleic acids, proteins, lipids, carbohydrates, glycans, glycoproteins, oligonucleotides, or combinations thereof.

[0111] Examples of protein detection methods include imaging mass spectrometry (MIBI), imaging mass cytometry (IMC), multiplex or singleplex immunohistochemistry, and other protein analysis methods performed in solution or in situ.

[0112] As spatial transcriptome methods, for example, multiplexed single-molecule in situ hybridization methods (e.g., MERFISH, seqFISH, osmFISH, Nanostring CosMx: Chen et al., Spatially Resolved, Highly Multiplexed RNA Profiling in Single Cells. Science 2015, 348(6233), aaa6090.; Shah et al., In Situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus. Neuron 2016, 92(2), 342-357; Lubeck et al., Single-Cell in Situ RNA Profiling by Sequential Hybridization. Nat Methods 2014, 11(4), 360-361.; Codeluppi et al., Spatial Organization of the Somatosensory Cortex Revealed by OsmFISH. Nat Methods 2018, 15 (11), 932-935; He et al., High-Plex Multiomic Analysis in FFPE at Subcellular Level by Spatial Molecular Imaging. bioRxiv January 2, 2022, p2021.11.03.467020), multiplexed in situ sequencing (e.g., STARMAP, ISS: Wang et al., Three-Dimensional Intact-Tissue Sequencing of Single-Cell Transcriptional States. Science 2018, 361(6400), eaat5691; Hilscher et al.,In Situ Sequencing: A High-Throughput, Multi-Targeted Gene Expression Profiling Technique for Cell Typing in Tissue Sections. In In Situ Hybridization Protocols; Nielsen, B.S., Jones, J., Eds.; Methods in Molecular Biology; Springer US: New York, NY, 2020; pp 313-329), and / or in situ RNA capture methods (e.g., ST / Visium, SLIDE-seq, HDST: Stahl et al., Tissue Sections by Spatial Transcriptomics. Science 2016, 353(6294), 78-82.; Rodriques et al., Slide-Seq: A Scalable Technology for Measuring Genome-Wide Expression at High Spatial Resolution. Science 2019, 363(6434), 1463-1467; Vickovic et al., High-Definition Spatial Transcriptomics for in Situ Tissue Profiling. Nat Methods 2019, 16(10), 987-990).

[0113] In one embodiment, the subsequent analysis method is multiplex fluorescence IHC that is performed before a further analysis method involving in situ RNA capture.

[0114] In some embodiments, the methods disclosed herein are (1) contacting the sample being tested for the presence of at least one target with at least one corresponding target-specific binding partner, wherein each target-specific binding partner of different specificities is linked to a different nucleic acid strand, said contacting (2) contacting the sample with a labeled imager strand having complementarity to a nucleic acid strand linked to the target-specific binding partner; (3) imaging the sample to detect the labeled bound imager strand; (4) optionally, removing the signal from the labeled bound imager strand; (5) optionally, detecting the location of at least one target in the sample by repeating at least once at least a part of steps (2) to (4) with a labeled imager strand having a unique composition as compared to at least one other labeled imager strand.

[0115] In some embodiments, any detectable label can be used to label the imager strand, and in some embodiments, this moiety is optically detectable.

[0116] In one embodiment, a fluorescent label is used. General categories of fluorescent labels include organic dyes, biological fluorophores, quantum dots, and nanoparticles including carbon dots. Specific fluorescent dyes include fluorescein, rhodamine, cyanine dyes, ALEXA dyes, DYLIGHT dyes, and ATTO dyes. In the examples herein, the use of four spectrally distinct fluorescent labels in a single detection is described. It is possible to use more than four spectrally overlapping fluorophores in a single detection. The use of software to assist in the detection of fluorophores having overlapping signals is known (see, e.g., US6,750,964). A variety of fluorescent dyes and filters are commercially available, and it is possible to practice the methods described herein using any practicable number of fluorescent labels. As described herein, in one embodiment, the method can be practiced using a single fluorescent label, two fluorescent labels, three fluorescent labels, four fluorescent labels, five fluorescent labels, six fluorescent labels, seven fluorescent labels, eight fluorescent labels, and eight or more fluorescent labels. Generally, when multiple fluorescent labels are used, the signals are detected in different detection channels corresponding to different regions of the optical spectrum. The following table shows four detection channels and representative fluorophores.

[0117]

Table 1

[0118] Additional methods of multiplex imaging for detecting multiple targets in the same sample are described in U.S. Patent No. 10,294,510, entitled "High-throughput and highly multiplexed imaging with programmable nucleic acid probes", U.S. Patent Application No. 2018 / 0164308, entitled "Methods for multiplex imaging using labeled nucleic acid imaging agents", U.S. Patent Application No. 2019 / 0376956, entitled "Multiplexed catalyzed reporter deposition", WO / 2021 / 007099, entitled "Improved Multiplexing Method", and WO / 2020 / 123961, entitled "Methods and Compositions For Sequentially Detecting Targets", the contents of each of which are incorporated herein by reference.

[0119] In some embodiments, the methods disclosed herein can result in a plurality of fluorescent images and corresponding brightfield morphological images obtained using H&E staining. In some embodiments, a control stain (e.g., DAPI nuclear stain) can be used to overlay the H&E-stained nuclei in the brightfield image with the fluorescent image.

[0120] In an imaging step, sometimes called a detection step, signals from histological staining, IHC staining, or in situ hybridization can be detected using a detection system. The nature of the detection system used can depend on the nature of the signal generator. The detection system can include a charge-coupled device (CCD) detection system, a fluorescence detection system, an electrical detection system, a photographic film detection system, a chemiluminescence detection system, an enzyme detection system, an optical detection system, a near-field detection system, or a total internal reflection (TIR) detection system.

[0121] F. Sample As used herein, the term "sample" means any natural or artificial biological fluid, cell, tissue, or fraction thereof, or other material that contains or is suspected of containing a target. A sample can be derived from a prokaryote or eukaryote, and thus can include, for example, cells from an animal, plant, or fungus. Thus, a sample can include or be derived from a specimen obtained from one or more individuals. In some embodiments, the sample contains cells. In some embodiments, the sample contains tissue.

[0122] In some embodiments, the sample is a tissue sample. As used herein, "tissue sample" means a series of cells obtained from an individual's tissue. The tissue can include nucleated cells containing chromosomal material. The source of the tissue sample can be fresh, frozen, FFPE, and / or solid tissue from a preserved organ or tissue sample, or a biopsy, or an aspirate, or blood or any blood component, or a body fluid such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, or cells from any point during the gestational period or development of the subject. The tissue sample can be primary or cultured cells or cell lines, or cultured tissue. The tissue sample can contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0123] In some embodiments, the tissue sample includes a tissue section. As used herein, a "section" of a tissue sample means a portion or piece of the tissue sample, e.g., a thin section of tissue or cells cut from the tissue sample. It is understood that multiple sections of a tissue sample can be subjected to analysis in accordance with the present invention. In some embodiments, a selected portion or section of the tissue includes a population of cells. In some embodiments, a selected portion or section of the tissue includes a homogeneous population of cells. In some embodiments, a selected portion or section of the tissue includes a heterogeneous population of cells. In some embodiments, the selected portion includes a region of the tissue, e.g., by way of non-limiting example, an organ, a tumor, stroma, a lumen. The selected portion or section may be as small as one cell or two cells, or may represent, for example, thousands of cells.

[0124] Any tissue sample from one or more individuals may be used. In one embodiment, the sample is tissue from an individual. In one embodiment, the sample is a tissue microarray sample comprising tissue from one or more individuals. Examples of tissue samples that may be used include, but are not limited to, breast, prostate, ovary, colon, lung, endometrium, stomach, salivary gland, or pancreas. The tissue sample can be obtained by various procedures including, but not limited to, surgical resection, aspiration, or biopsy.

[0125] The tissue may be fresh or frozen. In some embodiments, the tissue sample is a tissue section of a mammalian (e.g., human or mouse) brain, adrenal gland, colon, small intestine, stomach, heart, liver, skin, kidney, lung, pancreas, testis, ovary, prostate, uterus, thyroid, and spleen. The methods of the present disclosure can be used for any type of tissue, including, for example, cancerous tissue (including any cancer origin).

[0126] The sample used in the method described herein can be an unfixed or fixed biological sample. In some embodiments, the sample is fixed. Any fixative may be used. In one embodiment, the fixative is a solution containing an aldehyde. In some embodiments, the sample is fixed in a solution containing formalin. In some embodiments, the sample is paraffin-embedded. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

[0127] The tissue sample can be placed on a surface such as a slide, a flow cell, a 3D matrix, or a particle.

[0128] II. Kit In some embodiments, a kit for removing histological stains from a sample is disclosed. The kit includes (a) an acid agent having a pH from 1.0 to 4.0 and (b) a reducing agent having a pH from 8.0 to 10.0, as disclosed herein. The kit can further include a rinsing agent (e.g., deionized water) as disclosed herein. The kit can further include a staining reagent (e.g., an immunohistochemical reagent or an in situ fluorescence hybridization reagent) as disclosed herein. In some embodiments, agents (a) and (b) and optional (c) and (d) are separately contained in respective containers in the kit. The kit can include instructions for use.

[0129] In some embodiments, one or more of the foregoing methods can be automated and can be performed using an automated system. In some embodiments, all steps can be performed using an automated system.

[0130] III. Use The methods disclosed herein can be used for analytical, diagnostic, and therapeutic applications. In some embodiments, the methods disclosed herein can be used in imaging-based analysis, such as immunohistochemistry including chromogenic detection and immunofluorescent detection, or nucleic acid hybridization methods using, for example, fluorescent detection, or other methods. Analysis of samples from an individual by the methods described herein can be used diagnostically (e.g., to identify an individual having a particular disease, exposed to a particular toxin, or responsive to a particular therapeutic agent or organ transplant) and prophylactically (e.g., to identify an individual likely to develop a particular disease, responsive to a particular therapeutic agent, or accept a particular organ transplant). The methods disclosed herein can facilitate accurate and reliable analysis of multiple targets (e.g., disease markers) derived from the same sample.

Example

[0131] Example 1 This example illustrates a comparison of hematoxylin stain removal according to the present disclosure, comparing treatment with both an acid agent and a reducing agent to treatment with the acid agent alone or the reducing agent alone. Comparison with a positive control (no H&E staining) or a negative control (H&E staining without removal) is also shown.

[0132] Preparation of tissue slides: Human formalin-fixed paraffin-embedded (FFPE) tonsil tissue sections placed on slides (Amsbio LLC, Cambridge, MA) were first baked at 60 °C for 30 minutes. For H&E staining, the slides were processed on an Epredia Gemini automated slide stainer and stained with hematoxylin and eosin. Slides previously stained with H&E may also be used in this procedure.

[0133] Acid agent: An acid bath containing 1% HCl in 70% EtOH was prepared by mixing 6 mL of 10 N HCl with 500 mL of 70% denatured ethanol.

[0134] Reducing agent: A 1% NaBH4 aqueous solution adjusted to pH 9 with 1 M NaOH.

[0135] Similar results were observed using a 1% NaBH4 solution in 70% denatured ethanol (approx. 63% ethanol; 3 - 4% isopropanol and methanol) or 100% denatured ethanol (approx. 90% ethanol; 5% methanol, 5% isopropanol).

[0136] Eight tissue slides were processed according to the scheme listed in the following table.

[0137]

Table 2

[0138] For all slides applicable, the acid agent was brought into contact with the tissue for 2 minutes. For all slides applicable, the reducing agent was brought into contact with the tissue for 10 minutes.

[0139] Antigen retrieval was performed by incubating the slides in epitope retrieval solution 2 (AR9640, Leica Biosystems) at 100 °C for 20 minutes. The slides were then washed and subjected to immunostaining for four different targets (Ki67, PD1, CD3, CD68) using the FixVUE™ panel (Ultivue, Cambridge, MA) according to the manufacturer's manual. It should be noted that eosin was effectively removed during the antigen retrieval step without compromising subsequent immunostaining.

[0140] Figures 1A - 1C show the results of the analysis of the positive control (H&E staining not performed, slide number 01) and the negative control (H&E staining performed but hematoxylin removal not performed, slide number 02). Figure 1C shows the bright - field image of the negative control. As shown in Figure 1B, significantly less fluorescent signal was observed when hematoxylin removal was not performed. As shown in Figure 1C, the pattern of the remaining hematoxylin closely resembles the region with reduced fluorescence shown in Figure 1B.

[0141] Figures 2A - 2D show the fluorescence signals of tissues that did not undergo H&E staining and were untreated (Figure 2A, slide number 01), reductively treated (Figure 2B, slide number 03), acid - treated (Figure 2D, slide number 07), or treated with both (Figure 2C, slide #05). Figures 3A - 3D show the fluorescence signals of tissues that underwent H&E staining and were untreated (Figure 3A, slide number 02), reductively treated (Figure 3B, slide number 04), acid - treated (Figure 3D, slide number 08), or treated with both (Figure 3C, slide number 06).

[0142] Figures 4A - 8B show a comparison of fluorescence signals from pre - H&E - stained slides (slide number 06) that had H&E removed and were subsequently immunostained for Ki67, PD1, CD3, and CD68, and a positive control (without H&E staining, slide #01). As shown in the figures, the quality of immunostaining in each detection channel (Ki67, PD1, CD3, and CD68 respectively) was comparable to that of a positive control that had not been previously H&E - stained (Figures 5A, 6A, 7A, and 8A) when hematoxylin was removed according to the present disclosure (see Figures 5B, 6B, 7B, and 8B).

[0143] Example 2 This example illustrates the comparison of hematoxylin stain removal according to the present disclosure under different reducing agent conditions.

[0144] Cover slips were removed from human formalin - fixed paraffin - embedded (FFPE) tonsil tissue sections stained by a standard H&E protocol by immersing them in xylene for 2 days.

[0145] Next, the slides were rehydrated by incubating them in a series of graded alcohols (100%, 95%, 90%, 80%, water) for 1 minute. Next, the slides were incubated in a 1% (v / v) HCl solution in 70% ethanol for 2 minutes, and then the following was done.

[0146] 1. In 1% NaBH4 in 90% ethanol, 5% methanol, 5% isopropanol for 10 minutes (Figure 9A) In 1% NaBH4 in 2.90% ethanol, 5% methanol, 5% isopropanol for 5 minutes (Figure 9B) In 1% NaBH4 in 3.63% ethanol, 3% isopropanol, 3% methanol for 10 minutes (Figure 9C) In 1% NaBH4 in 4.63% ethanol, 3% isopropanol, 3% methanol for 5 minutes (Figure 9D) In 1% NaBH4 in carbonate-bicarbonate buffer at pH 9.2 for 10 minutes (Figure 9E) In 1% NaBH4 in carbonate-bicarbonate buffer at pH 9.2 for 5 minutes (Figure 9F)

[0147] After these different treatments, a coverslip was attached to the slide and bright-field microscope images were taken. Figures 9A - 9F show that hematoxylin was removed according to the present disclosure.

[0148] Equivalents The foregoing description is considered to be sufficient to enable one of ordinary skill in the art to practice the embodiments. In the foregoing description and examples, specific embodiments are described in detail, which are illustrative of the best mode contemplated by the inventors. However, regardless of how detailed the foregoing may be expressed in the document, the embodiments may be practiced in many ways and are to be construed in accordance with the appended claims and any equivalents thereof.

[0149] As used herein, the term "about" refers to numerical values, including, for example, integers, fractions, and percentages, whether explicitly indicated or not. The term "about" generally refers to a range of numerical values (e.g., + / - 5 - 10% of the recited range) that one of ordinary skill in the art would consider to be equivalent to the recited value (e.g., those having the same function or result). When terms such as "at least" and "about" precede a list of numerical values or ranges, those terms modify all of the values or ranges provided in the list. In some instances, the term "about" may include a numerical value rounded to the nearest significant digit.

[0150] The embodiments of the present invention will be described in detail, but it should be understood that other embodiments are contemplated. Therefore, the present invention is not intended to be limited to the details of the configurations and the arrangements of the components described in the following description or shown in the drawings. The present invention can have other embodiments and can be implemented or carried out in various ways. Also, when describing the embodiments, specific terms are relied on for clarity.

[0151] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include the plural referents unless the context clearly dictates otherwise. For example, references to a sheet or portion are also intended to include the manufacture of multiple sheets or portions. References to a sheet including "a" component are intended to include other components in addition to the specified component.

[0152] Also, when describing the embodiments, specific terms are relied on for clarity. Each term is intended to have its broadest meaning as understood by those skilled in the art and to include all technical equivalents that operate in a similar manner to achieve a similar purpose.

[0153] Ranges can be expressed herein as from "about" or "approximately" one particular value and / or to "about" or "approximately" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value.

[0154] "Comprising", "containing", or "including" means that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other such compounds, materials, particles, method steps, even if they have the same function as those named.

[0155] It should also be understood that a reference to one or more method steps does not exclude the presence of additional method steps, or method steps intervening between those steps that are explicitly identified. Similarly, a reference to one or more components in an organization or system should be understood not to exclude the presence of additional components, or components intervening between those components that are explicitly identified.

Claims

1. A method for removing histological staining from a histologically stained sample, comprising: (a) contacting the sample with an acid agent having a pH from 1.0 to 4.0; and (b) contacting the sample with a reducing agent having a pH from 8.0 to 10.0, thereby removing the histological staining from the histologically stained sample.

2. A method for preparing a histologically stained sample for a subsequent analysis method for detecting at least one target, comprising: (a) contacting the sample with an acid agent having a pH from 1.0 to 4.0; (b) contacting the sample with a reducing agent having a pH from 8.0 to 10.0; (c) contacting the sample with a staining agent to stain at least one target in the sample; and (d) optionally, detecting the at least one target in the sample.

3. A method for removing histological staining from a histologically stained sample, comprising: (a) contacting the sample with an acid agent having a pH from 1.0 to 4.0; (b) removing the acid agent; (c) contacting the sample with a reducing agent having a pH from 8.0 to 10.0, thereby removing the histological staining from the histologically stained sample.

4. A method for preparing a histologically stained sample for a subsequent analysis method for detecting at least one target, comprising: (a) contacting the sample with an acid agent having a pH from 1.0 to 4.0; (b) removing the acid agent; (c) contacting the sample with a reducing agent having a pH from 8.0 to 10.0; (d) removing the reducing agent; (e) contacting the sample with a staining agent to specifically stain at least one target in the sample; and (f) optionally, detecting the at least one target in the sample.

5. The method according to claim 1 or 2, wherein the acid agent is removed from the sample.

6. The method according to any one of claims 1 to 4, wherein the acid agent is removed from the sample using a rinsing agent.

7. The method according to claim 1 or 2, wherein the reducing agent is removed from the sample.

8. The method according to any one of claims 1 to 4, wherein the reducing agent is removed from the sample using a rinsing agent.

9. The method according to any one of claims 1 to 4, wherein the histological staining contains hematoxylin.

10. The method according to claim 9, wherein the histological staining further contains eosin.

11. The method according to claim 10, further comprising detecting hematoxylin and eosin staining before step (a).

12. The method according to any one of claims 1 to 4, wherein the pH of the acidic agent is from 1.0 to 2.

0.

13. The method according to any one of claims 1 to 4, wherein the acidic agent contains hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, hydroiodic acid, sulfamic acid, perchloric acid, or a combination thereof.

14. The method according to claim 13, wherein the acidic agent contains hydrochloric acid (HCl).

15. The method according to any one of claims 1 to 4, wherein the acidic agent contains a solvent selected from water, ethylene glycol, polyethylene glycol, propylene glycol, ethanol, methanol, or a combination thereof.

16. The method according to claim 14, wherein the acidic agent contains 1% HCl in 70% ethanol.

17. The method according to any one of claims 1 to 4, wherein the pH of the reducing agent is from 8.0 to 9.

0.

18. The method according to any one of claims 1 to 4, wherein the reducing agent contains a reducing agent selected from sodium borohydride, sodium cyanoborohydride, potassium bromate, sodium sulfite, sodium dithionite, sodium thiosulfate, sodium bisulfite, sodium triethylborohydride, and sodium triacetoxyborohydride, or a combination thereof.

19. The method according to any one of claims 1 to 4, wherein the reducing agent contains a solvent selected from water, alcohol, ethylene glycol, and propylene glycol, or a combination thereof.

20. The method according to claim 19, wherein the solvent of the reducing agent contains ethanol.

21. The method according to claim 18, wherein the reducing agent contains 1% sodium borohydride in water.

22. The step of contacting the sample with the acidifying agent is performed for 20 minutes, 15, 10, 5, 4, 3, 2, or 1 minute or less, and / or the step of contacting the sample with the reducing agent is performed for 20 minutes, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 minutes or less, the method according to any one of claims 1 to 4.

23. During the step of contacting the sample with the acidifying agent, the temperature of the sample is maintained between 20°C and 30°C, and / or during the step of contacting the sample with the reducing agent, the temperature of the sample is maintained between 20°C and 30°C, the method according to any one of claims 1 to 4.

24. Further including applying heat to the sample so that the sample and the acidifying agent are maintained at a predetermined temperature during the step of contacting the sample with the acidifying agent, and / or further including applying heat to the sample so that the sample and the reducing agent are maintained at a predetermined temperature during the step of contacting the sample with the reducing agent, the method according to any one of claims 1 to 4.

25. Furthermore: (1) Contacting a tissue sample that has been tested for the presence of at least one target with at least one corresponding target-specific binding partner, wherein each target-specific binding partner with different specificities is linked to a different nucleic acid strand, said contacting; (2) Contacting the tissue sample with a labeled imaging strand having complementarity to the nucleic acid strand linked to the target-specific binding partner; (3) Imaging the sample to detect the labeled binding imaging strand; (4) Optionally, repeating at least a part of steps (2) to (3) at least once with a labeled imaging strand having a unique composition in comparison with at least one other labeled imaging strand; including thereby detecting the location of the at least one target in the sample, the method according to any one of claims 1 to 4.

26. The method according to any one of claims 1 to 4, wherein the sample contains cells or tissues.

27. A kit for removing histological stains from a tissue sample, comprising: (a) An acidifying agent having a pH from 1.0 to 4.0; (b) A reducing agent having a pH from 8.0 to 10.0; (c) Optionally, a rinsing agent; (d) Optionally, a staining reagent and, The kit comprising.

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