Non-destructive antigen retrieval method for three-dimensional histology under mild conditions

Abstract

A novel method for antigen retrieval in formalin-fixed paraffin-embedded (FFPE) tissue samples for three-dimensional immunohistochemistry (3D IHC). The method utilizes an aqueous chemical solution containing a detergent and a dissociation agent for antigen retrieval. The detergent contains a zwitterionic substance, and the dissociation agent contains a water-soluble boron cluster compound. This method allows for the treatment of tissue samples with the aqueous chemical solution under mild heating conditions to achieve optimal antigen retrieval for subsequent 3D IHC.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 787,230, filed April 11, 2025, the entire contents of which, including any figures, tables or drawings, are incorporated herein by reference. Background Technology

[0002] After 30 years of development, antigen retrieval technology has had a tremendous impact on global histological research and has transformed traditional two-dimensional histology based on tissue sections. [1,2] Antigen retrieval technology enables reliable immunohistochemical (IHC) detection of archived samples, particularly suitable for tissues stored in formalin-fixed paraffin-embedded (FFPE) tissue blocks. IHC allows visualization of specific biomolecules within tissues known as antigens. The morphology and location of these antigens are crucial for understanding tissue biology, improving diagnostic accuracy, classifying diseases, and making treatment decisions based on detected biomarkers.

[0003] Furthermore, IHC is preferably suitable for FFPE sections, as the thinner sections yield better morphological characteristics, enabling high-resolution observation of tissues embedded using this method. In addition, FFPE is the preferred method for tissue preservation because the tissue is dried and fixed in a solid block, remaining stable at room temperature without refrigeration, and avoiding hazards or leakage problems during preservation and transportation.

[0004] The development of reliable antigen retrieval techniques has greatly promoted the application of IHC in FFPE sections. As the name suggests, antigen retrieval restores tissue antigens, facilitating detection using IHC. Histological analysis of FFPE sections without antigen retrieval results in poor IHC signals and nonspecific staining, producing low-quality sections and images unsuitable for histological diagnosis. Conversely, with antigen retrieval-assisted IHC, pathologists can directly observe the spatial distribution and location of different antigens in archived or freshly prepared FFPE sections, obtaining the optimal morphological quality required for diagnosis. This method has spawned a multi-billion dollar industry, with every standard histopathology diagnostic laboratory utilizing antigen retrieval-assisted IHC in its daily operations. Today, using antigen retrieval-assisted IHC to detect tissue biomarkers has become a standard diagnostic method for all cancers and other diseases. While two-dimensional histology has achieved great success, progress in the three-dimensional analysis of histological samples has been minimal. Three-dimensional histology provides a true view of tissue morphology and cell distribution. However, deep-penetrating IHC lacks reliability for archived samples preserved in paraffin. In traditional two-dimensional histology, antigen retrieval techniques for paraffin sections have made revolutionary progress, but these methods cannot be extended to three-dimensional histology. Therefore, for retrospective and future studies of three-dimensional histological samples, there is an urgent need for a novel and reliable antigen retrieval method suitable for three-dimensional histology. [3] . Summary of the Invention

[0005] This article discloses a method for repairing antigens from FFPE tissue blocks for three-dimensional immunohistochemistry (3D IHC). In an embodiment, the method includes deparaffinizing, washing, and rehydrating the tissue, and applying an aqueous chemical solution under mild conditions (below about 80°C) for a duration that varies from about 10 minutes to about 2 weeks, depending on the specific properties of the sample.

[0006] In some embodiments, paraffin is first removed from the tissue using organic solvent A. Then, the tissue is washed with organic solvent B to remove organic solvent A and dissolved paraffin. Next, the tissue is rehydrated by a series of washes in a methanol-water solution with increasing water content. Finally, the aqueous chemical solution is applied to the tissue for antigen retrieval, followed by 3D IHC.

[0007] In some embodiments, the aqueous chemical solution comprises a surfactant and a dissociating agent. In some embodiments, the surfactant comprises a zwitterionic substance, such as alkyl sulfonate betaine. In a preferred embodiment, the alkyl sulfonate betaine is N-hexadecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate. In some embodiments, the dissociating agent is a water-soluble boron cluster compound. In a preferred embodiment, the boron cluster compound is a dodecylhydro-closed dodecoborate ion. Attached Figure Description

[0008] This patent or application document contains at least one color-drawn drawing. A copy of this patent or patent application publication with the color drawing will be provided by the Patent Office upon request and after payment of the relevant fees.

[0009] Figure 1 This illustrates antigen retrieval from a batch of formalin-fixed human colon tissue. The top row (control group) was not paraffin-embedded and underwent direct 3D IHC, while the bottom two rows were treated with FFPE. The second row did not undergo antigen retrieval, while the third row underwent antigen retrieval using the method of this invention. Red indicates indirect 3D IHC using rabbit anti-NaK-ATPase antibody. Green indicates cells labeled with exogenous lectin using wheat germ lectin. The "Merged" column shows the overlapping portions of the two staining methods.

[0010] Figure 2 The effect of adding a dissociative agent to detergent under mild heating conditions of 55°C on antigen retrieval is illustrated. Vimentin staining (magenta) shows a significant reduction in nonspecific staining.

[0011] Figure 3 A magnified view shows the improved staining quality achieved with the addition of dodecoborate in antigen retrieval. Experimental replication results are also shown. Detailed Implementation

[0012] Selected definition Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used herein are intended to include the plural forms. Furthermore, with regard to “including,” “includes,” “having,” “has,” “with,” or variations thereof as used in the specific description and / or claims, such terms are intended to be non-exclusive in a manner similar to the term “comprising.” The transitional terms / phrases (and any grammatical variations thereof) “comprising,” “comprises,” “comprise,” “consisting essentially of,” “consists essentially of,” “consisting,” and “consists” are used interchangeably.

[0013] The phrase "consisting essentially of" or "consists essentially of" indicates that the claim covers embodiments that include the specified materials or steps, as well as embodiments that do not substantially affect the essential and novel features of the claim.

[0014] The term "about" refers to an acceptable range of error for a specific value, as determined by a person skilled in the art, which depends in part on the method of measurement of the value, i.e., the limitations of the measurement system. In the case of compositions containing an ingredient content and using the term "about," these compositions contain the ingredient at said content, which varies around the value by 0-10% (error range) (X±10%). In other cases, the term "about" provides for a variation of 0-10% around a given value (error range) (X±10%). Clearly, this variation represents a range within 10% above or below the given value, for example, X±1%, X±2%, X±3%, X±4%, X±5%, X±6%, X±7%, X±8%, X±9%, or X±10%.

[0015] In this disclosure, to avoid listing and describing every and all values ​​within a range in detail, ranges are represented in abbreviated form. Where appropriate, any suitable value within the range may be chosen as the upper limit, lower limit, or endpoint of the range. For example, a range of 0.1–1.0 represents the endpoint values ​​0.1 and 1.0, and the intermediate values ​​0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges covered by the range of 0.1–1.0, such as 0.2–0.5, 0.2–0.8, 0.7–1.0, etc. Values ​​within a range are envisioned to have at least two significant digits; for example, a range of 5–10 indicates all values ​​between 5.0 and 10.00 and between 5.00 and 10.00, including endpoint values. When using ranges herein, combinations and sub-combinations of ranges (e.g., sub-ranges within the disclosed range) and specific embodiments thereof are explicitly included.

[0016] "Reduction" means a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0017] "Increase" means a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0018] As used herein, the terms “determine,” “measure,” “evaluate,” and “verify” are used interchangeably and include both quantitative and qualitative determinations.

[0019] Antigen retrieval methods for 3D IHC This invention relates to an antigen retrieval method suitable for FFPE tissue blocks for subsequent 3D IHC. In one aspect, the method includes the following main steps: 1. The tissue was deparaffinized using organic solvent A to dissolve the paraffin and extract it from the tissue matrix; 2. Perform a transitional wash on the tissue to remove organic solvent B and dissolved paraffin, and to prevent paraffin from redepositing into the tissue; 3. The tissue is rehydrated using a gradient solvent to change the tissue from a dehydrated state to a hydrated state; 4. Antigen retrieval of the tissue using an aqueous solution containing a surfactant and a dissociative agent under mild heating conditions to restore protein antigenic epitopes; and 5. The tissue was then subjected to subsequent 3D IHC.

[0020] In a preferred embodiment, the size of the tissue block should not exceed approximately 3 cm × approximately 3 cm × approximately 5 mm. In some embodiments, the individual or total size of the tissue block may exceed the aforementioned approximately 3 cm × approximately 3 cm × approximately 5 mm specification by approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or higher.

[0021] In some embodiments, the dewaxing step uses organic solvent A to rapidly and completely dissolve the paraffin embedded in the tissue. In some embodiments, organic solvent A includes, but is not limited to, xylene, toluene, and limonene. In a preferred embodiment, organic solvent A comprises low-boiling-point alkanes. In some embodiments, organic solvent A comprises saturated straight-chain alkanes and saturated cyclic alkanes, or mixtures thereof, with boiling points above 37°C. In a more preferred embodiment, organic solvent A comprises saturated cyclic alkanes. In an even more preferred embodiment, organic solvent A is cyclohexane or methylcyclopentane.

[0022] In one embodiment, the transitional washing step utilizes organic solvent B, a solvent mixture miscible with both the dewaxing organic solvent A and the subsequent gradient solvents used for rehydration. This solvent mixture is used to efficiently extract paraffin from the tissue. In some embodiments, solvent B includes, but is not limited to, a mixture of tetrahydrofuran and methanol, or alternatively, an ether, ketone, or alcohol having alkyl and aromatic side chains. In a preferred embodiment, solvent B comprises a mixture of tetrahydrofuran and methanol.

[0023] In some embodiments, the rehydration treatment step includes sequentially placing the tissue in a series of solutions with increasing proportions of water for displacement. In some embodiments, the solutions include, but are not limited to, methanol-water mixtures with decreasing methanol concentrations. Optionally, in some embodiments, soluble additives may be added to promote tissue rehydration, such as surfactants S, organic and inorganic salts, acid-base regulators, or other organic solvents. In some embodiments, the surfactants S include, but are not limited to, zwitterionic surfactants, nonionic surfactants, anionic surfactants, or cationic surfactants.

[0024] In this embodiment, the antigen retrieval step includes applying an aqueous detergent solution containing a surfactant and a dissociative agent to the tissue to promote global refolding of proteins throughout the tissue. Without being bound by any theory, reversal of formalin crosslinks is auxiliary, but insufficient, for achieving antigen retrieval against IHC. Due to wax embedding, the tissue changes from a hydrated state to a hydrophobic matrix-embedded state. This results in most proteins undergoing "inside-out" folding, where hydrophobic residues that were originally embedded inside are exposed, while hydrophilic residues that would normally serve as antibody binding targets are embedded inside. Thus, heat treatment or protease digestion in two-dimensional histology can help reverse the protein refolding process in an aqueous environment, and the energy barrier for refolding is lower if formalin crosslinks are removed or if the protein is digested by proteases. In this case, surfactants can have the same thermodynamic promoting effect on the refolding of hydrophobically exposed proteins in the presence of a dissociative agent. In some embodiments, additional optional additives (such as water-miscible organic solvents, salts, pH buffers, ion chelating agents, and cyclodextrins) can further promote this process.

[0025] In some embodiments, the surfactant comprises an alkylated sulfobetaine reagent, said alkylated sulfobetaine reagent reacting with a closed dodecoborate ion ([B...) as a dissociating agent...) 12 H 12 ] 2- Coupling. In a preferred embodiment, the general formula structure of the surfactant shown in Formula 1 is:

[0026] Formula 1 Where n ranges from 0 to 10, R 1 Independently selected from: straight-chain alkyl, branched alkyl, cyclic alkyl, alkyl containing steroidal groups, and aromatic alkyl. In a preferred embodiment, n=3, wherein R 1 Including but not limited to: straight-chain hexadecyl, tetradecyl, dodecyl, decyl, or octadecyl. In other embodiments, it may be replaced with compounds having more variable R. 1 Other derivatives of the group, which depend on their properties as surfactants (or active agents). In one embodiment, the surfactant shown in Formula 2 has the following structure:

[0027] Formula 2 In this implementation, the hydroxyl group between the quaternary ammonium group and the sulfonic acid group can be omitted without negatively impacting the antigen retrieval process in 3D histology. In another example, the characteristics of the zwitterionic head can differ, as illustrated in the following example of a zwitterionic surfactant shown in Formula 3:

[0028] Formula 3 In another example, electrically neutral zwitterionic surfactants can be used for antigen retrieval in 3D histology. In an embodiment, the zwitterionic surfactant has an X-L1-Y-L2-Z structure, wherein X is a hydrophobic molecule independently selected from alkyl and aromatic chains, and optionally modified with functional groups, such as N-heterocyclic, olefinic, or alkyne groups; wherein Y is a cationic group independently selected from quaternary ammonium, pyridinium, imidazolium, triazolium, pyrrolonnium, tetrazolium, and quaternary phosphonium groups; wherein Z is an anionic group independently selected from sulfonate, carboxylate, phosphonate, phosphonite, sulfinate, sulfate, and borate groups; and wherein L1 is an optional linker between X and Y, independently selected from alkyl, olefinic, and alkyne chains. L1 and L2 are chained alkyl chains, hydroxylated alkyl chains, aromatic chains, and aminolated alkyl chains; wherein L2 is a linker between Y and Z, and the linker is independently selected from: alkyl chains, alkene chains, hydroxylated alkyl chains, aromatic chains, and aminolated alkyl chains, and wherein the alkyl groups of L1 and L2 can be straight-chain, branched, or cyclic.

[0029] In some embodiments, the ionizing agent may be selected from Hofmeister sequence compounds exhibiting ionizing properties. In some embodiments, the ionizing agent includes, but is not limited to: urea, guanidine salts, perchlorates, thiocyanates, iodides, triiodides, perrhenates, bromides, ferricyanide (III) ions, ferrocyanide (II) ions, hexafluorophosphates, borates, tetrafluoroborates, boron cluster compounds, and polyoxometalates of vanadium, molybdenum, and tungsten.

[0030] In some embodiments, the boron cluster compound may be a derivative of an anionic boron cluster containing 10-20 boron atoms, optionally comprising 0-4 non-boron atoms, such as carbon (referred to as carborane salts) or metal ions (e.g., bis(7,8-dicarboborane)cobalt). Boron atoms may be bonded to hydrogen atoms or another atom to form derivatives, wherein different vertices on the boron cluster backbone may be fully and / or independently derivatized. Hydrogen atoms bonded to boron atoms may be substituted, for example, with chlorine, bromine, iodine, methyl, ethyl, trifluoromethyl, hydroxyl, methoxy, ethoxy, alkoxy, amino, cyano, cyanate, thiocyanate, isocyanate, isothiocyanate, etc.

[0031] In a preferred embodiment, the boron cluster has 12 boron atoms and contains no other non-boron atoms, and is also referred to as closed dodecoborate. The general formula of closed dodecoborate is:

[0032] In this embodiment, boron atoms are represented by spheres, each of which may bear different independent substituents. In a preferred embodiment, all vertices are substituted with hydrogen, i.e., dodecylhydro-closed dodecylborate, and the substituents on each sphere include, but are not limited to, groups of BH, BF, BCl, BBr, BOH, B-CH3, and B-NH2. The counter cation of the closed dodecylborate can vary independently with the soluble salt, such as lithium, sodium, potassium, rubidium, cesium, calcium, magnesium, hydrogen, tris(hydroxymethyl)methylammonium, ethylenediammonium, ammonium, methylammonium, hydrazine, ethylammonium, triethanolamine, etc. In a preferred embodiment, the counter cation is sodium.

[0033] In some embodiments, the concentration range of the zwitterionic surfactant is from about 0.001 M to about 0.5 M. Any concentration range or concentration between 0.001 M and 0.5 M can be used. In a preferred embodiment, the concentration of the zwitterionic surfactant is about 0.1 M.

[0034] In embodiments, the concentration of the dodecylhydro-closed dodecylborate ion can be independently in the range of 0.001 M to 0.5 M. Any concentration range or concentration between 0.001 M and 0.5 M can be used. In a preferred embodiment, the concentration of the dodecylhydro-closed dodecylborate ion is 0.25 M.

[0035] In some embodiments, the incubation temperature for the antigen retrieval step ranges from about 30°C to about 80°C. In some embodiments, the incubation temperature for the antigen retrieval step ranges from about 55°C to about 60°C. In a more preferred embodiment, the incubation temperature for the antigen retrieval step is about 55°C.

[0036] In some embodiments, the incubation time for the antigen retrieval step ranges from about 10 minutes to about 2 weeks. In a preferred embodiment, the incubation time for the antigen retrieval step is equal to or less than 72 hours.

[0037] In another embodiment, the aqueous solution used for antigen retrieval optionally includes additives, such as organic solvents, to further dissolve proteins and residual paraffin. In some embodiments, the organic solvent includes, but is not limited to: water-miscible solvents, such as alcohols, ketones, ethers, esters, nitriles, amides, and polar aprotic solvents. In some examples, the organic solvent includes, but is not limited to: methanol, ethanol, isopropanol, 1,2-hexanediol, ethylene glycol, glycerol, propylene glycol, ethylene carbonate, propylene carbonate, acetone, butanone, tetrahydrofuran, 1,4-dioxane, dimethyl sulfoxide, acetonitrile, dimethylacetamide, dimethylformamide, N-methylpyrrolidone, dimethylimidazolium ketone, dimethylacrylurea, hexamethylphosphoramide, pyridine, sulfolane, methanesulfonylmethane; salts, such as water-soluble inorganic salts; pH adjusters, such as acids, bases, such as buffers; and ion chelating agents for removing non-target cations that may alter the ionic strength of the antigen retrieval solution and for preventing nucleic acid damage. In other examples, the organic solvents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid, phosphonic acid, and sulfonic acid.

[0038] Materials and Methods For antigen retrieval in two-dimensional tissue sections, the first step is always deparaffinization, which is the process of removing solid wax from the sections. This step is usually accomplished by washing the tissue in xylene or limonene, followed by gradient rehydration in a series of decreasing ethanol solutions, and then antigen retrieval.

[0039] However, even with a series of optimizations, combinations, and extended processing times, this method is only applicable to FFPE sections and not to tissue volumes extracted from wax. The quality and resulting images from 3D IHC still lack specificity, with weak or no signal.

[0040] The failure of deparaffinization and antigen retrieval based on 2D sections is attributed to the failure to consider the tissue volume effect, which complicates the penetration and removal of chemicals within the tissue. Furthermore, while 2D histology has the advantage of using signal amplification techniques to significantly enhance contrast, this is inefficient and unsuitable for 3D histology. 2D antigen retrieval methods fail to fully address the core issue of enabling sensitive antibody detection of antigens. Finally, the advantage of 2D antigen retrieval lies in the ability to optimize various antigen retrieval methods and conditions using a small number of sections, whereas antigen retrieval in 3D histology is essentially a one-off event, as any damage can lead to complete tissue failure. This further hinders the use of tissue heating methods (typically above 80°C), protease digestion, and denaturing detergent treatments, which have been so successful in 2D histology.

[0041] The following is an exemplary protocol for retrieval of antigens from FFPE samples for 3D IHC analysis: (i) At room temperature (rt), the tissue was washed with pure cyclohexane or methylcyclopentane to remove paraffin; (ii) Wash the tissue 0-3 times with cyclohexane or methylcyclopentane to ensure adequate deparaffin removal; (iii) Wash the tissue with 85% (v / v) tetrahydrofuran (THF) and 15% (v / v) methanol (MeOH) at room temperature; (iv) Wash the tissue 0-3 times with THF:MeOH at a volume ratio of 85:15 to ensure complete removal of cyclohexane or methylcyclopentane; (v) At room temperature, tissues were rehydrated in pure water by a gradient methanol washing step (ranging from 100% MeOH to 0% MeOH). (vi) Wash the tissue with pure water; (vii) The tissue is placed in a mixture of antigen retrieval solutions and, depending on the tissue size, incubated at 37°C to 55°C for 1 hour to 2 days. The antigen retrieval solution is a pure aqueous solution containing 0.1M Zwittergent® zwitterionic detergent (n-alkyl-N,N-dimethyl-3-ammonium-1-propanesulfonate) and 0.25M dodecylhydro-closed dodecylborate ions. (viii) Depending on the size of the tissue, wash the tissue thoroughly with pure water at a temperature of 37°C to 55°C for 1 minute to 1 hour, 0 to 3 times. (ix) Next, 3D IHC was performed on the tissue.

[0042] All patents, patent applications, provisional applications and publications mentioned or cited herein are incorporated herein by reference in their entirety (including all figures and tables), but only to the extent that they do not contradict the express teachings of this specification.

[0043] The following are illustrative examples of processes used to practice the present invention. These examples should not be construed as limiting. Unless otherwise stated, all percentages are by weight and all solvent mixture proportions are by volume.

[0044] Example 1—3D IHC Analysis of Antigen Retrieval from FFPE Samples Compared with FFPE samples that underwent only deparaffin treatment, the addition of water-based antigen retrieval solution significantly improved the quality of 3DIHC. Figure 1 A pair of samples is shown, in which control tissue (formalin-fixed human colon tissue) is preserved in PBS (without FFPE treatment); and Figure 2Paired samples from the same tissue batch are shown. These samples were paraffin-embedded, deparaffinized, and stained with 3D IHC with and without antigen retrieval methods. Tissues retrieval using the method of this invention exhibited excellent staining quality, showing no significant difference from the first control group not embedded in paraffin.

[0045] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art can make various modifications or alterations based thereon, which should be included within the spirit and scope of this application and the scope of the appended claims. Furthermore, any element or limitation of any invention or implementation thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or implementation thereof, and all such combinations are conceived within the scope of this invention without being limited thereto.

[0046] Exemplary Implementation Implementation Method 1. A method for antigen retrieval in formalin-fixed paraffin-embedded (FFPE) tissues for three-dimensional immunohistochemistry (3D IHC), the method comprising: (a) Obtain FFPE tissue; (b) The tissue was deparaffinized using organic solvent A; (c) Wash the tissue with organic solvent B; (d) The tissue was rehydrated using a series of gradient solvent solutions; (e) Wash the tissue with pure water; (f) The tissue was placed in an aqueous detergent solution for antigen retrieval; (g) Wash the tissue with pure water; and (h) Next, 3D IHC was performed on the tissue. The organic solvent A used to deparaffinize the tissue is selected from: saturated cyclic alkanes, and mixtures of saturated straight-chain alkanes and saturated cyclic alkanes; The organic solvent B used to wash the tissue is selected from a mixture of tetrahydrofuran and methanol. The gradient solvent solution used for rehydration of the tissue comprises a methanol-water mixture with decreasing methanol concentration, and optionally one or more additives; and The aqueous detergent solution used to repair the antigen contains surfactants, a release agent, and optional additives.

[0047] Implementation Method 2. According to the method of Implementation Method 1, wherein the gradient solvent solution optionally contains one or more additives selected from: surfactant S, organic salt, inorganic salt, acid-base control agent and organic solvent, wherein the surfactant S is selected from: zwitterionic surfactant, nonionic surfactant, anionic surfactant and cationic surfactant.

[0048] Implementation Method 3. The method according to Implementation Method 1 or 2, wherein the aqueous detergent solution optionally contains an additive selected from: water-miscible solvents, salts, pH adjusters, ion chelating agents, and cyclodextrins.

[0049] Embodiment 4. The method according to any one of the foregoing embodiments, wherein the surfactant comprises alkyl sulfonate betaine, the alkyl sulfonate betaine being reacted with a surfactant of the formula [B... 12 H 12 ] 2- The closed-loop dodecoborate ion coupling, wherein the surfactant has the structure shown in Formula 1:

[0050] Formula 1, Where n ranges from 0 to 10; where R 1 Independently selected from: straight-chain alkyl, branched-chain alkyl, cyclic alkyl, steroidal group and aromatic alkyl, or wherein n=3, wherein R 1 Selected from: straight-chain hexadecyl, tetradecyl, dodecyl, decyl and octadecyl.

[0051] Embodiment 5. The method according to any one of the foregoing embodiments, wherein the surfactant has the structure shown in Formula 2:

[0052] Equation 2.

[0053] Embodiment 6. The method according to any one of Embodiments 1 to 4, wherein the surfactant has the structure shown in Formula 3:

[0054] Formula 3.

[0055] Embodiment 7. The method according to any one of Embodiments 1 to 4, wherein the surfactant is an amphoteric surfactant having an X-L1-Y-L2-Z structure. Wherein, X is a hydrophobic group selected from: alkyl, aromatic chains, and optional functional group modifications, which are selected from the group consisting of: hydroxyl, ether, phenyl, N-heterocyclic, alkenyl, and alkynyl. Wherein, Y is a cationic group, which is independently selected from: quaternary ammonium, pyridinium, imidazolylium, triazolium, pyrrolomium, tetrazolium, and quaternary phosphorus; Wherein, Z is an anionic group, which is independently selected from: sulfonate, carboxylate, phosphonate, phosphonite, sulfite, sulfate and borate; Wherein, L1 is an optional linker between X and Y, which is independently selected from: alkyl, alkene, hydroxylated alkyl, aromatic chain and aminolated alkyl chain; Wherein, L2 is an optional linker between Y and Z, which is independently selected from: alkyl, alkene, hydroxylated alkyl, aromatic chain, and aminolated alkyl; and The alkyl groups of L1 and L2 are selected from straight-chain alkyl groups, branched alkyl groups, and cyclic alkyl groups.

[0056] Embodiment 8. The method according to any one of the preceding embodiments, wherein the alkylated sulfobetaine is N-hexadecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate, wherein the concentration of N-hexadecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate is about 0.1M.

[0057] Implementation Method 9. The method according to any one of the preceding embodiments, wherein the ionizing agent is selected from: urea, guanidine salts, perchlorate, thiocyanate, iodide, triiodide, perrhenate, bromide, ferricyanide (III) ion, ferrocyanide (II) ion, hexafluorophosphate, borate, tetrafluoroborate, and polyoxometalates of vanadium, molybdenum, and tungsten.

[0058] Embodiment 10. The method according to any one of the foregoing embodiments, wherein the liquid release agent is a boron cluster compound. The boron cluster compound is a derivative of an anionic boron cluster ion containing 10-20 boron atoms, optionally comprising 0-4 non-boron atoms selected from the group consisting of carbon and metal ions, wherein the boron atoms are attached to hydrogen atoms or substituted atoms to form the derivative, the substituted atoms being selected from carbon, nitrogen, oxygen, sulfur, chlorine, bromine, and iodine; and The apex of the boron cluster is either partially or completely derivatized, wherein the derivatizing group is independently selected from: chlorine, bromine, iodine, methyl, ethyl, trifluoromethyl, hydroxyl, methoxy, ethoxy, alkoxy, amino, cyano, cyanate, thiocyanate, isocyanate, and isothiocyanate.

[0059] Embodiment 11. The method according to Embodiment 10, wherein the boron cluster compound is a closed dodecoborate having Formula 4:

[0060] Equation 4, Each vertex is independently selected from: BH, BF, BCl, BI, BOH, B-CH3, and B-NH3.

[0061] Embodiment 12. The method according to Embodiment 11, wherein the boron cluster compound is dodecylhydro-closed dodecylborate, wherein the counter cation of the closed dodecylborate is independently selected from: lithium, sodium, potassium, rubidium, cesium, calcium, magnesium, hydrogen, tris(hydroxymethyl)methylammonium, ethylenediamine, ammonium, methylammonium, hydrazine, ethylammonium and triethanolamine.

[0062] Embodiment 13. The method according to Embodiment 12, wherein the counter cation of the closed dodecoborate is sodium, and wherein the concentration of sodium in the closed dodecoborate is about 0.25M.

[0063] Embodiment 14. The method according to any one of the foregoing embodiments, wherein the concentration range of the zwitterionic surfactant is from about 0.001M to about 0.5M.

[0064] Embodiment 15. The method according to any one of the foregoing embodiments, wherein the concentration of the dodecylhydro-closed dodecylborate ion is in the range of about 0.001M to about 0.5M.

[0065] Implementation Method 16. The method according to any one of the foregoing embodiments, wherein the incubation temperature range for the antigen retrieval step is from about 30°C to about 80°C.

[0066] Implementation 17. The method according to Implementation 16, wherein the incubation temperature range for the antigen retrieval step is from about 45°C to about 60°C.

[0067] Implementation Method 18. The method according to Implementation Method 17, wherein the incubation temperature for the antigen retrieval step is about 55°C.

[0068] Implementation Method 19. The method according to any one of the foregoing embodiments, wherein the incubation time for the antigen retrieval step ranges from about 10 minutes to about 2 weeks.

[0069] Implementation 20. The method according to Implementation 19, wherein the incubation time for the antigen retrieval step is approximately 72 hours.

[0070] References 1. Shi SR et al. Immunohistochemistry of antigen retrieval: A review and future prospect of more than 20 years of research and diagnosis. Journal of Histochemistry and Cytochemistry, January 2011; 59 (1):13-32. doi: 10.1369 / jhc.2010.957191. PMID: 21339172; PMCID: PMC3201121. 2. Shi SR et al. Antigen retrieval technology: Current research progress. Journal of Histochemistry and Cytochemistry (official journal of the Society for Histochemistry), 2001; Vol. 49, No. 8: 931-937. doi:10.1177 / 002215540104900801. 3. Yau CN et al. INSIHGT: An easy-to-use multi-scale, multimodal 3D space biology platform. Nature Communications, 2024, Vol. 15, 10888. doi.org / 10.1038 / s41467-024-55248-0.

Claims

1. A method for antigen retrieval in FFPE tissue for three-dimensional immunohistochemistry, the method comprising: (a) Obtain FFPE tissue; (b) The tissue was deparaffinized using organic solvent A; (c) Wash the tissue with organic solvent B; (d) Rehydration treatment of the tissue with a series of gradient solvent solutions; (e) Wash the tissue with pure water; (f) Antigen retrieval is performed by placing the tissue in an aqueous detergent solution; (g) Wash the tissue with pure water; as well as (h) Next, three-dimensional immunohistochemistry was performed on the tissue. Wherein, the organic solvent A used to deparaffinize the tissue is selected from: saturated cyclic alkanes, and mixtures of saturated straight-chain alkanes and saturated cyclic alkanes; Wherein, the organic solvent B used for washing the tissue is selected from: a mixture of tetrahydrofuran and methanol; The gradient solvent solution used for rehydrating the tissue comprises a methanol-water mixture with decreasing methanol concentration, and optionally one or more additives; and The aqueous detergent solution used for antigen retrieval includes a surfactant, a release agent, and optional additives.

2. The method according to claim 1, wherein, The gradient solvent solution optionally includes one or more additives selected from: surfactant S, organic salts, inorganic salts, acid-base control agents, and organic solvents, wherein the surfactant S is selected from: zwitterionic surfactants, nonionic surfactants, anionic surfactants, and cationic surfactants.

3. The method according to claim 1, wherein, The aqueous detergent solution contains additives selected from: water-miscible solvents, salts, pH adjusters, ion chelating agents, and cyclodextrins.

4. The method according to claim 1, wherein, The surfactant comprises alkylated sulfonated betaine, which is in combination with the formula [B 12 H 12 ] 2- The closed-loop dodecoborate ion coupling, wherein the surfactant has the structure shown in Formula 1: Formula 1, Where n ranges from 0 to 10; where R 1 Independently selected from: straight-chain alkyl, branched-chain alkyl, cyclic alkyl, steroidal group and aromatic alkyl, or wherein n=3, wherein R 1 Selected from: straight-chain hexadecyl, tetradecyl, dodecyl, decyl and octadecyl.

5. The method according to claim 4, wherein, The surfactant has the structure shown in Formula 2: Equation 2.

6. The method according to claim 4, wherein, The surfactant has the structure shown in Formula 3: Formula 3.

7. The method according to claim 4, wherein, The surfactant is an amphoteric surfactant, wherein the amphoteric surfactant has an X-L1-Y-L2-Z structure. Wherein, X is a hydrophobic group, which is selected from alkyl, aromatic chains, and optional functional group modifications, wherein the functional group modifications are selected from hydroxyl, ether, phenyl, N-heterocyclic, alkenyl, and alkynyl. Wherein, Y is a cationic group, and the cationic group is independently selected from: quaternary ammonium, pyridinium, imidazolylium, triazolium, pyrrolomium, tetrazolium and quaternary phosphorus; Wherein, Z is an anionic group, and the anionic group is independently selected from: sulfonate, carboxylate, phosphonate, phosphonite, sulfite, sulfate and borate; Wherein, L1 is an optional linker between X and Y, and the linker is independently selected from: alkyl, alkene, hydroxylated alkyl, aromatic chain and aminolated alkyl chain; Wherein, L2 is an optional linker between Y and Z, and the linker is independently selected from: alkyl, alkene, hydroxylated alkyl, aromatic chain, and aminolated alkyl; and The alkyl groups of L1 and L2 are selected from straight-chain alkyl groups, branched alkyl groups, and cyclic alkyl groups.

8. The method according to claim 4, wherein, The alkylated sulfobetaine is N-hexadecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate, wherein the concentration of the N-hexadecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate is about 0.1 M.

9. The method according to claim 4, wherein, The ionizing agent is selected from: urea, guanidine salts, perchlorates, thiocyanates, iodides, triiodides, perrhenates, bromides, ferricyanide (III) ions, ferrocyanide (II) ions, hexafluorophosphates, borates, tetrafluoroborates, and polyoxometalates of vanadium, molybdenum, and tungsten.

10. The method of claim 4, wherein, The liquid-dissolving agent is a boron cluster compound. The boron cluster compound is a derivative of anionic boron cluster ions containing 10-20 boron atoms, optionally comprising 0-4 non-boron atoms selected from the group consisting of carbon and metal ions, wherein the boron atoms are attached to hydrogen atoms or substituted atoms to form the derivative, and the substituted atoms are selected from carbon, nitrogen, oxygen, sulfur, chlorine, bromine, and iodine; and The apex of the boron cluster is either partially or completely derivatized, wherein the derivatizing group is independently selected from: chlorine, bromine, iodine, methyl, ethyl, trifluoromethyl, hydroxyl, methoxy, ethoxy, alkoxy, amino, cyano, cyanate, thiocyanate, isocyanate, and isothiocyanate.

11. The method according to claim 4, wherein, The boron cluster compound is a closed dodecoborate having Formula 4: Equation 4, Each vertex is independently selected from: BH, BF, BCl, BI, BOH, B-CH3, and B-NH3.

12. The method according to claim 11, wherein, The boron cluster compound is a dodecylhydro-closed dodecylborate, wherein the counter cation of the closed dodecylborate is independently selected from: lithium, sodium, potassium, rubidium, cesium, calcium, magnesium, hydrogen, tris(hydroxymethyl)methylammonium, ethylenediamine, ammonium, methylammonium, hydrazine, ethylammonium, and triethanolamine.

13. The method according to claim 12, wherein, The counter cation of the closed dodecoborate is sodium, wherein the concentration of sodium in the closed dodecoborate is about 0.25 M.

14. The method according to claim 7, wherein, The concentration of the zwitterionic surfactant ranges from about 0.001 M to about 0.5 M.

15. The method according to claim 7, wherein, The concentration of dodecahydro-closed dodecaborate ions ranges from about 0.001 M to about 0.5 M.

16. The method according to claim 1, wherein, The incubation temperature range for the antigen retrieval step is from about 30°C to about 80°C.

17. The method according to claim 16, wherein, The incubation temperature used in the antigen retrieval step is in the range of about 55°C to about 60°C.

18. The method according to claim 17, wherein, The incubation temperature used in the antigen retrieval step is approximately 55°C.

19. The method according to claim 1, wherein, The incubation time for the antigen retrieval step ranges from about 10 minutes to about 2 weeks.

20. The method according to claim 19, wherein, The incubation time for the antigen retrieval step is approximately 72 hours.

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