CN114486423A - Preparation method of compound liquid for paraffin tissue section dewaxing repair blocking - Google Patents
Preparation method of compound liquid for paraffin tissue section dewaxing repair blocking Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention relates to a preparation method of a compound liquid for paraffin tissue section dewaxing repair blocking, which can be used for paraffin tissue section dewaxing, antigen repair and endogenous peroxidase blocking under the heating condition, and the tissue section after treatment can be immediately subjected to immunohistochemical staining. The composite liquid has the same dewaxing effect as xylene at the temperature of 72-75 ℃, is nontoxic, nonvolatile and tasteless, and is an environment-friendly aqueous dewaxing agent for incubation; under the heating condition of 95-100 ℃, the dewaxing effect is completed at one time, the antigen repairing effect is performed, the blocking effect of endogenous peroxidase is completed at the same time, and the paraffin tissue section after the heat treatment can be subjected to immunohistochemical staining after being rinsed. Dewaxing, antigen restoration and blocking are completed at one time, and the method has the advantages of simplicity, convenience, short experimental operation time, difficulty in falling, environmental friendliness, no toxicity and the like.
Description
Technical Field
The invention relates to pathological immunohistochemical paraffin section pretreatment, belongs to the technical field of pathological detection reagents, and particularly relates to a preparation method of a compound liquid for paraffin tissue section dewaxing repair blocking.
Background
Immunohistochemistry (IHC) is an indispensable important technical means in the current pathological diagnosis process, and plays an immeasurable role in the aspects of tumor diagnosis, typing identification, medication guidance, prognosis evaluation, curative effect evaluation and the like. Pathological paraffin sections are the most common and major test samples for Immunohistochemistry (IHC). The sample is a paraffin-embedded tissue section, is water-insoluble and cannot be directly used for immunohistochemical detection, and the sample needs to be pretreated before immunohistochemical detection, namely, the sample needs to be subjected to dewaxing, hydration, antigen retrieval and endogenous peroxidase blocking treatment. The typical paraffin tissue section pretreatment basic steps are as follows: dewaxing xylene (or an environment-friendly dewaxing agent), performing ethanol gradient hydration (such as absolute ethanol, 95% ethanol, 85% ethanol and distilled water), repairing antigen, rinsing, blocking and rinsing again. After a series of operation processes, the immunohistochemical staining reaction of the paraffin section can be carried out.
The classical paraffin tissue section dewaxing, hydration, repair and blocking are complex in operation steps, xylene is often used for dewaxing, the xylene has certain toxicity and is not environment-friendly, and certain damage can be brought to operators. Replacing classical dewaxing, hydration, repair and blockage is a necessary trend in technology development.
Disclosure of Invention
The invention relates to preparation of a compound liquid (high pH) for paraffin tissue section dewaxing repair blocking and application thereof, which can be used for paraffin tissue section dewaxing, antigen repair and endogenous peroxidase blocking under the heating condition, and the tissue section after treatment can be immediately subjected to immunohistochemical staining. The composite liquid comprises the following components: aqueous wax dissolver, tissue penetrant, dispersant, defoaming agent, tissue protective agent, blocking agent, high pH value repair solution (containing Triton X-100, EDTA, Tris and the like) and the like. The composite liquid has the same dewaxing effect as xylene at the temperature of 72-75 ℃, is nontoxic, nonvolatile and tasteless, and is an environment-friendly aqueous dewaxing agent for incubation; under the heating condition of 95-100 ℃, the effects of dewaxing and antigen repair are completed at one time, meanwhile, the blocking effect of endogenous peroxidase can be achieved, and the paraffin tissue section after heat treatment can be subjected to immunohistochemical staining after being rinsed. Dewaxing, antigen restoration and blocking are completed at one time, and the method has the advantages of simplicity, convenience, short experimental operation time, difficulty in falling, environmental friendliness, no toxicity and the like.
The invention provides a preparation method and application of a compound liquid for paraffin tissue section dewaxing repair blocking.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a compound liquid for paraffin tissue section dewaxing repair blocking comprises the following steps:
s1: preparing an aqueous dewaxing agent;
s2: preparing a repairing liquid;
s3: preparing an endogenous peroxidase blocking agent;
s4: preparing a composite liquid.
Preferably, in S1, the aqueous dewaxing agent is prepared by mixing a water-soluble dewaxing agent, a tissue penetrating agent, a dispersing agent, an antifoaming agent, and distilled water.
Preferably, the water-soluble dewaxing agent is formed by mixing 0.25-2.5% of PEG200, 0.05-0.75% of ethylene glycol, 0.25-1.00% of tetrahydrofuran-2-methanol and 0.05-0.85% of fatty alcohol-polyoxyethylene ether; the invention utilizes the mixture composed of PEG200, glycol, tetrahydrofuran-2-methanol, fatty alcohol polyoxyethylene ether and the like, has good solubility in water phase, and can dissolve paraffin under the condition of heating; the tissue penetrating agent is formed by mixing 0.1-0.75% of Triton X-100 and 0.01-0.5% of glycerol; triton X-100 and glycerol, or at least one of them, are used in the present invention.
Preferably, the dispersant is formed by mixing 0.1-0.75% of Triton X-100 and 0.01-0.05% of Tween-20; the dispersant is surfactant such as Tween-20, Triton X-100, or at least one compound; the defoaming agent is formed by mixing 0.01-0.1% of ethylene glycol tertiary butyl ether and 0.03-0.05% of polyoxypropylene glycerol ether; ethylene glycol tert-butyl ether and polyoxypropylene glycerol ether, two or at least one compound in the prepared dewaxing repair blocking composite liquid.
Preferably, in S2, the repair solution is prepared from a buffer solution, a tissue penetrating agent and a metal chelating agent.
Preferably, the buffer is 0.01-0.05mM Tris-HCl and the pH of the buffer is 9.0; the buffer solution is a main component in the repair solution and is prepared from Tris hydrochloric acid; the metal chelating agent is EDTA of 0.001-0.002 mM; interference of metal ions can be reduced; the tissue penetrating agent is prepared by mixing 0.01-0.05% of glycerol and 0.1-0.75% of Triton X-100.
Preferably, in S3, the endogenous peroxidase blocker is prepared from a reducing agent; wherein the reducing agent is 0.1-0.5% disodium stannous citrate; the activity of peroxidase within the tissue can be inactivated.
Preferably, in S4, a complex liquid is prepared using an aqueous dewaxing agent, a repair liquid, and an endogenous peroxidase blocking agent.
Preferably, the composite liquid has dewaxing effect and the dewaxing steps are as follows: dewaxing is accomplished by incubation at 72-75 ℃. Namely: baking the paraffin tissue slices in an oven at 60-65 ℃ for 30-60 minutes, then putting the slices into a composite liquid preheated to 72-75 ℃, and soaking for 20 minutes at constant temperature; taking out the slices, directly placing the slices into another cylinder, preheating the slices to a composite liquid at 72-75 ℃, soaking the slices for 20 minutes at a constant temperature, and then washing the slices with water to finish dewaxing; the operation is not carried out in a boiling water environment, and antigen repair and endogenous peroxidase blocking are required to be carried out to dye IHC; when the composite liquid is used for pure dewaxing operation, the heating dewaxing incubation temperature is 72-75 ℃; the heating dewaxing incubation time is 20 minutes, and the soaking is carried out twice at constant temperature; after the heating dewaxing is finished, the rinsing water is tap water or PBS buffer solution.
Preferably, the dewaxing and antigen retrieval steps are: under the condition of heating at 95-100 deg.C, dewaxing and antigen repairing action can be completed at one time. Namely: placing the tissue paraffin section in an oven at 60-65 deg.C, baking for 30-60 min, placing the section in a composite liquid preheated to 72-75 deg.C, and heating the composite liquid to 95-100 deg.C for 20 min; taking out the slices, rinsing the slices once in a composite liquid preheated to 72-75 ℃, and then washing the slices with water to finish dewaxing repair; in the subsequent operation, endogenous peroxidase can be blocked according to the conventional operation and then used for IHC dyeing, when the compound liquid is used for dewaxing and repairing operation, the compound liquid needs to be heated to 72-75 ℃ in advance, and the paraffin tissue section cannot be directly put into the cold compound liquid, so that paraffin on the section is prevented from being adhered to the slide and is not beneficial to the diffusion of the dissolved wax and the wax; after the paraffin tissue section is placed, the compound liquid is continuously heated to 95-100 ℃, so that the dewaxing can be promoted and the purpose of antigen restoration can be achieved at the same time; after the paraffin tissue slices are placed, the compound liquid needs to be continuously heated to 95-100 ℃ for dewaxing repair, and the duration time needs 20 minutes; after the paraffin tissue section is dewaxed and repaired, the paraffin tissue section is taken out and rinsed once by using the compound liquid preheated to 72-75 ℃, and then can be rinsed by using tap water or distilled water.
Preferably, the steps of dewaxing, antigen retrieval and endogenous peroxidase blocking are: the complex liquid contains blocker when in use, and dewaxing, antigen repairing and endogenous peroxidase blocking effects are completed at one time under the condition of heating at 95-100 ℃. Namely: placing the tissue paraffin section in an oven at 60-65 deg.C, baking for 30-60 min, placing the section in a composite liquid preheated to 72-75 deg.C, and heating the composite liquid to 95-100 deg.C for 20 min; taking out the slices, rinsing the slices once in a composite liquid preheated to 72-75 ℃, and then washing the slices with water to finish the processes of dewaxing, repairing, blocking and the like; the paraffin section can be directly used for IHC staining, the paraffin tissue section subjected to dewaxing repair blocking is finished when the complex liquid is subjected to dewaxing repair blocking operation, the paraffin tissue section is taken out and rinsed once by the complex liquid preheated to 72-75 ℃, and then the paraffin tissue section is washed by tap water or distilled water, so that the blocking effect of endogenous peroxidase can be finished without additional operation.
Compared with the prior art, the invention has the beneficial effects that:
the chemical reagents used by the dewaxing repair blocking composite liquid are nontoxic, nonvolatile and free of pungent smell, and after the defoaming agent is added, the liquid reagents can be effectively prevented from volatilizing to generate irritant gas in a heating state, and meanwhile, the dewaxing repair effect is prevented from being influenced by bubbles formed on the surface of a glass slide. The dewaxing repair blocking composite liquid has basically the same dewaxing effect as xylene, has no influence on the health of operators, and does not produce toxic waste liquid. The dewaxing repair liquid is used for a pathological IHC detection technology, does not damage a histiocyte structure, effective components in histiocytes and tissue antigens in the using process, and meets the requirement of high-quality staining sheets required by clinical pathological diagnosis and research.
The dewaxing repair blocking compound liquid has good detection effect on pathological IHC, antigen repair and blocking of endogenous peroxidase are carried out while dewaxing is carried out, and dewaxing, hydration, repair and blocking are finished at one time, so that the operation steps are shortened, and the experiment time is saved.
Drawings
FIG. 1 is a flow chart of a method for preparing a compound liquid for paraffin tissue section dewaxing repair blocking according to the present invention;
FIG. 2 is a graph of the HE staining result of the compound fluid for repairing and blocking paraffin tissue section by dewaxing according to the present invention;
FIG. 3 is a graph showing the IHC staining results (lung cancer, TTF-1) of a complex fluid preparation method for paraffin tissue section deparaffinization repair block according to the present invention;
FIG. 4 is a graph of the HE staining result of the compound fluid for repairing and blocking paraffin tissue section by dewaxing according to the present invention;
FIG. 5 is a graph showing the result of IHC staining (tonsil, CK 5/6) by a complex liquid preparation method for paraffin tissue section deparaffinization repair block according to the present invention;
FIG. 6 is a graph of the HE staining result of the compound fluid for repairing and blocking paraffin tissue section by dewaxing according to the present invention;
FIG. 7 is a chart of IHC staining results (tonsil, p 63) of a complex liquid preparation method for paraffin tissue section deparaffinization repair blocking provided by the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example one
Referring to fig. 1, a method for preparing a complex liquid for paraffin tissue section dewaxing repair blocking includes the following steps:
s1: preparing an aqueous dewaxing agent;
s2: preparing a repairing liquid;
s3: preparing an endogenous peroxidase blocking agent;
s4: preparing a composite liquid.
In this embodiment, in S1, the aqueous dewaxing agent is prepared by mixing a water-soluble dewaxing agent, a tissue penetrating agent, a dispersing agent, an antifoaming agent, and distilled water.
In this embodiment, the water-soluble dewaxing agent is formed by mixing 0.5% of PEG200, 0.5% of ethylene glycol, 0.55% of tetrahydrofuran-2-methanol and 0.15% of fatty alcohol-polyoxyethylene ether; the tissue penetrating agent is formed by mixing 0.1% of Triton X-100 and 0.25% of glycerol.
In the embodiment, the dispersant is 0.01 percent of Tween-20; the antifoaming agent was 0.02% ethylene glycol tertiary butyl ether.
In this embodiment, in S2, the repair solution is prepared from a buffer solution, a tissue penetrating agent, and a metal chelating agent.
In this example, the buffer was Tris-HCl 0.01mM, and the pH of the buffer was 9.0; the metal chelator is 0.001mM EDTA; the tissue penetrating agent is formed by mixing 0.1% of Triton X-100 and 0.25% of glycerol.
In this embodiment, in S3, the endogenous peroxidase blocking agent is prepared from a reducing agent; wherein the reducing agent is 0.3 percent of disodium stannous citrate.
In this example, in S4, a complex solution is prepared using an aqueous dewaxing agent, a repair solution, and an endogenous peroxidase blocking agent.
Preparation operation step 1: preparing 0.01mM Tris-HCl (pH9.0) buffer solution, and adding EDTA to make the concentration reach 0.001mM, thus obtaining the antigen repairing solution. Weighing 1.21g of Tris and 0.29g of EDTA, dissolving in 1L of distilled water, and adjusting the pH value to 9.0 by using hydrochloric acid after full dissolution.
Preparation operation step 2: and (2) sequentially weighing water-soluble dewaxing agents (PEG 200, ethylene glycol, fatty alcohol-polyoxyethylene ether, tetrahydrofuran-2-methanol), penetrating agents (Triton X-100 and glycerol), dispersing agents (Tween-20) and defoaming agents (ethylene glycol tert-butyl ether) according to the composition proportion, adding the water-soluble dewaxing agents, the ethylene glycol, the fatty alcohol-polyoxyethylene ether and the tetrahydrofuran-2-methanol into the antigen retrieval liquid obtained in the step (1), and fully stirring and dissolving the mixture. The solution is the composite liquid A.
Preparation operation step 3: and preparing a 3% disodium stannous citrate aqueous solution by using purified water to obtain a compound liquid B.
Preparation operation step 4: when the compound liquid is used for clinical application, the liquid B is added into the liquid A, and the final concentration of the liquid B is 0.3%.
In this example, the compounded liquid thereof functions as a dewaxing agent (hydration) in an immunohistochemical experiment: baking the tissue paraffin section in an oven at 60-65 ℃ for 60 minutes, then putting the section into the composite liquid preheated to 75 ℃, and soaking for 20 minutes at constant temperature; taking out the slices, directly placing the slices into another cylinder, preheating the slices to 75 ℃ in the composite liquid, soaking the slices for 20 minutes at constant temperature, and then washing the slices with water to finish dewaxing and hydration. Dewaxed hydrated pathological sections were HE stained, dehydrated, cleared and mounted.
Referring to fig. 2, the results show that no wax is left on the surface of the section, the section is dewaxed cleanly, the tissue cell morphology is complete, the staining is clear and obvious, and the pathological section treated by the reagent has good dewaxing effect and does not damage the cell structure of the pathological tissue.
In this example, the prepared complex liquid has dewaxing, hydration, antigen retrieval and endogenous peroxidase blocking effects in immunohistochemical experiments: baking the tissue paraffin section in an oven at 60-65 ℃ for 60 minutes, then putting the section into the composite liquid preheated to 75 ℃, and continuously heating the composite liquid to 100 ℃ for 20 minutes; taking out the slices, rinsing the slices once in a composite liquid preheated to 75 ℃, and then flushing the slices with water to finish dewaxing repair blocking;
referring to fig. 3, the result of staining the lung cancer tissue with TTF-1 shows that the wax on the surface of the section is not obviously retained, the dewaxing effect is obviously clean, good antigen repair can be seen by staining with TTF-1, the tissue cell morphology is kept in a good original state and the integrity thereof, the cell nucleus hematoxylin negatively expressed by TTF-1 is clearly and obviously stained, and the signal of positive staining of TTF-1 is clear and definite. The compound liquid meets the requirements of deparaffinization, hydration, antigen restoration and blocking of immunohistochemistry of tissue slices.
Example two
Referring to fig. 1, a method for preparing a complex liquid for paraffin tissue section dewaxing repair blocking includes the following steps:
s1: preparing an aqueous dewaxing agent;
s2: preparing a repairing liquid;
s3: preparing an endogenous peroxidase blocking agent;
s4: preparing a composite liquid.
In this embodiment, in S1, the aqueous dewaxing agent is prepared by mixing a water-soluble dewaxing agent, a tissue penetrating agent, a dispersing agent, an antifoaming agent, and distilled water.
In this embodiment, the water-soluble dewaxing agent is formed by mixing 2.5% of PEG200, 0.25% of ethylene glycol, 0.75% of tetrahydrofuran-2-methanol and 0.05% of fatty alcohol-polyoxyethylene ether; the tissue penetrating agent is formed by mixing 0.05 percent of Triton X-100 and 0.25 percent of glycerol.
In the embodiment, the dispersant is 0.01 percent of Tween-20; the antifoaming agent was 0.05% polyoxypropylene glyceryl ether.
In this embodiment, in S2, the repair solution is prepared from a buffer solution, a tissue penetrating agent, and a metal chelating agent.
In this example, the buffer was Tris-HCl 0.01mM, and the pH of the buffer was 9.0; the metal chelator is 0.001mM EDTA; the tissue penetrating agent is formed by mixing 0.05 percent of Triton X-100 and 0.25 percent of glycerol.
In this embodiment, in S3, the endogenous peroxidase blocking agent is prepared from a reducing agent; wherein the reducing agent is 0.3 percent of disodium stannous citrate.
In this example, in S4, a complex solution is prepared using an aqueous dewaxing agent, a repair solution, and an endogenous peroxidase blocking agent.
Preparation operation step 1: preparing 0.01mM Tris-HCl (pH9.0) buffer solution, and adding EDTA to make its concentration reach 0.001mM, thus obtaining the antigen retrieval solution. Weighing 1.21g of Tris and 0.29g of EDTA, dissolving in 1L of distilled water, and adjusting the pH value to 9.0 by using hydrochloric acid after full dissolution.
Preparation operation step 2: and (2) sequentially weighing water-soluble dewaxing agents (PEG 200, ethylene glycol, tetrahydrofuran-2-methanol and fatty alcohol polyoxyethylene ether), penetrating agents (Triton X-100 and glycerol), dispersing agents (Tween-20) and defoaming agents (polyoxypropylene glycerol ether) according to the composition proportion, adding the water-soluble dewaxing agents, the ethylene glycol, the tetrahydrofuran-2-methanol and the fatty alcohol polyoxyethylene ether into the antigen retrieval solution obtained in the step (1), and fully stirring and dissolving the mixture. The solution is the composite liquid A.
Preparation operation step 3: and preparing a 3% disodium stannous citrate aqueous solution by using purified water to obtain a compound liquid B.
Preparation operation step 4: when the compound solution is used, the solution B is added into the solution A, and the final concentration of the solution B is 0.3 percent.
In this example, the compounded liquid thereof functions as a dewaxing agent (hydration) in an immunohistochemical experiment: baking the tissue paraffin section in an oven at 60-65 ℃ for 60 minutes, then putting the section into the composite liquid preheated to 75 ℃, and soaking for 20 minutes at constant temperature; taking out the slices, directly placing the slices into another cylinder, preheating the slices to 75 ℃ in the composite liquid, soaking the slices for 20 minutes at constant temperature, and then washing the slices with water to finish dewaxing and hydration. Deparaffinizing the hydrated pathological sections, HE staining, dehydrating, clearing and mounting.
Referring to fig. 4, the results show that no wax is left on the surface of the section, the section is dewaxed cleanly, the tissue cell morphology is complete, the staining is clear and obvious, and the pathological section treated by the reagent has good dewaxing effect and does not damage the cell structure of the pathological tissue.
In this example, the prepared complex liquid has dewaxing, hydrating and antigen retrieval effects in immunohistochemical experiments: baking the tissue paraffin section in an oven at 60-65 ℃ for 60 minutes, then putting the section into the composite liquid preheated to 75 ℃, and continuously heating the composite liquid to 100 ℃ for 20 minutes; and taking out the slices, rinsing the slices once in the composite liquid preheated to 75 ℃, and then flushing the slices with water to finish dewaxing repair blocking.
Referring to fig. 5, the result of CK5/6 staining on tonsil tissues shows that the wax on the surface of the section is not obviously retained, the dewaxing effect is obviously clean, good antigen repair can be seen through CK5/6 staining, the form of tissue cells is kept in a good original state and the integrity, the staining of CK5/6 negative expression cell nucleus hematoxylin is clear and obvious, and the signal of CK5/6 positive staining is clear and definite. The dewaxing repair liquid meets the requirements of immunohistochemical dewaxing, hydration and antigen repair of tissue slices.
EXAMPLE III
Referring to fig. 1, a method for preparing a complex liquid for paraffin tissue section dewaxing repair blocking includes the following steps:
s1: preparing an aqueous dewaxing agent;
s2: preparing a repairing liquid;
s3: preparing an endogenous peroxidase blocking agent;
s4: preparing a composite liquid.
In this embodiment, in S1, the aqueous dewaxing agent is prepared by mixing a water-soluble dewaxing agent, a tissue penetrating agent, a dispersing agent, an antifoaming agent, and distilled water.
In this embodiment, the water-soluble dewaxing agent is formed by mixing 2.5% of PEG200, 0.5% of ethylene glycol and 0.65% of fatty alcohol-polyoxyethylene ether; the tissue penetrating agent is composed of 0.1% of Triton X-100 and 0.25% of glycerol.
In the embodiment, the dispersant is 0.01 percent of Tween-20; the antifoaming agent is 0.02% of ethylene glycol tertiary butyl ether and 0.05% of polyoxypropylene glycerol ether.
In this embodiment, in S2, the repair solution is prepared from a buffer solution, a tissue penetrating agent, and a metal chelating agent.
In this example, the buffer was Tris-HCl 0.01mM, and the pH of the buffer was 9.0; the metal chelator is 0.001mM EDTA; the tissue penetrating agent is formed by mixing 0.1% of Triton X-100 and 0.25% of glycerol.
Preparation operation step 1: preparing 0.01mM Tris-HCl (pH9.0) buffer solution, and adding EDTA to make its concentration reach 0.001mM, thus obtaining the antigen retrieval solution. Weighing 1.21g of Tris and 1.29g of EDTA0, dissolving in 1L of distilled water, fully dissolving, and adjusting the pH value to 9.0 by using hydrochloric acid.
Preparation operation step 2: and (2) sequentially weighing water-soluble dewaxing agents (PEG 200, ethylene glycol and fatty alcohol polyoxyethylene ether), penetrating agents (Triton X-100 and glycerol), dispersing agents (Tween-20) and defoaming agents (ethylene glycol tert-butyl ether and polyoxypropylene glycerol ether) according to the composition proportion, adding the water-soluble dewaxing agents, the penetrating agents and the glycerol into the antigen retrieval solution obtained in the step (1), and fully stirring and dissolving the mixture.
In this example, the dewaxing repair solution served as a dewaxing agent (hydration) in immunohistochemical experiments: placing the tissue paraffin section in an oven at 60-65 ℃ for baking for 60 minutes, then placing the section in dewaxing repair liquid preheated to 75 ℃, and soaking for 20 minutes at constant temperature; taking out the slices, directly putting the slices into another cylinder, preheating the slices to 75 ℃ in advance, soaking the slices for 20 minutes at constant temperature, and then washing the slices with water to finish dewaxing and hydration. Deparaffinizing the hydrated pathological sections, HE staining, dehydrating, clearing and mounting.
Referring to fig. 6, the results show that no wax is left on the surface of the section, the section is dewaxed cleanly, the tissue cell morphology is complete, the staining is clear and obvious, and the pathological section treated by the reagent has good dewaxing effect and does not damage the cell structure of the pathological tissue.
In this example, the dewaxing repair liquid has dewaxing, hydrating and antigen repairing effects in an immunohistochemical experiment: placing the tissue paraffin section in an oven at 60-65 ℃ for baking for 60 minutes, then placing the section in a dewaxing repair liquid preheated to 75 ℃, and continuously heating the dewaxing repair liquid to 100 ℃ for 20 minutes; and after the slices are taken out, the slices are rinsed once in dewaxing repair liquid which is preheated to 75 ℃, and then the slices are rinsed by water to finish dewaxing repair.
Referring to fig. 7, the staining result of p63 on tonsil tissues shows that the wax on the surface of the section is not obviously retained, the dewaxing effect is obviously clean, good antigen repair can be seen by p63 staining, the tissue cell morphology is kept in a good original state and the integrity, the staining of the cell nucleus hematoxylin expressed negatively by p63 is clear and obvious, and the signal of the positive staining of p63 is clear and definite. The dewaxing repair liquid meets the requirements of immunohistochemical dewaxing, hydration and antigen repair of tissue slices.
The dewaxing repair blocking compound liquid of the invention comprises the following basic steps of pretreatment of paraffin tissue sections: heating dewaxing repair blocking compound liquid is placed into the slices to complete dewaxing repair blocking effect at one time, and antigen-antibody immune reaction immunohistochemical staining can be performed after rinsing. The key steps of dewaxing, hydration, antigen repair and endogenous peroxidase blocking are completed at one time through the treatment of the four-in-one dewaxing repair liquid, and the test proves that the dewaxing hydration repair blocking effect of the compound is completely consistent with that of a classical method, and the compound can be applied to clinical pathological immunohistochemical detection.
The dewaxing repair blocking compound liquid integrates the operation steps of section dewaxing, hydration, antigen repair and blocking, and completes three classic steps in one step, and has the advantages that: the dewaxing repair blocking composite liquid is non-toxic and not easy to volatilize, does not generate pungent smell in the operation process, has good biodegradability, and has the same dewaxing effect as dimethyl benzene.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. A preparation method of a compound liquid for paraffin tissue section dewaxing repair blocking is characterized by comprising the following steps:
s1: preparing an aqueous dewaxing agent;
s2: preparing a repairing liquid;
s3: preparing an endogenous peroxidase blocking agent;
s4: preparing a composite liquid.
2. The method as claimed in claim 1, wherein the aqueous dewaxing agent is prepared by mixing a water-soluble dewaxing agent, a tissue penetrating agent, a dispersing agent, an antifoaming agent, and distilled water in S1.
3. The method for preparing a complex liquid for paraffin tissue section dewaxing repair block according to claim 2, wherein the water-soluble dewaxing agent is composed of 0.25-2.5% of PEG200, 0.05-0.75% of ethylene glycol, 0.25-1.00% of tetrahydrofuran-2-methanol and 0.05-0.85% of fatty alcohol-polyoxyethylene ether by mixing; the tissue penetrating agent is prepared by mixing 0.1-0.75% of Triton X-100 and 0.01-0.5% of glycerol.
4. The method for preparing the compound liquid for repairing the block of paraffin tissue section by dewaxing according to claim 2, wherein the dispersant is composed of 0.1-0.75% of Triton X-100 and 0.01-0.05% of Tween-20; the defoaming agent is formed by mixing 0.01-0.1% of ethylene glycol tertiary butyl ether and 0.03-0.05% of polyoxypropylene glycerol ether.
5. The method as claimed in claim 1, wherein the repair solution is prepared from a buffer solution, a tissue penetrating agent and a metal chelating agent in S2.
6. The method for preparing a complex liquid for paraffin tissue section deparaffinization repair block according to claim 5, wherein the buffer is 0.01-0.05mM Tris-HCl and the pH of the buffer is 9.0; the metal chelating agent is EDTA of 0.001-0.002 mM; the tissue penetrating agent is prepared by mixing 0.01-0.05% of glycerol and 0.1-0.75% of Triton X-100.
7. The method for preparing the complex liquid for paraffin tissue section deparaffinization repair block according to claim 1, wherein in S3, the endogenous peroxidase blocking agent is prepared from a reducing agent; wherein the reducing agent is 0.1-0.5% disodium stannous citrate.
8. The method of claim 1, wherein the S4 is performed by using an aqueous dewaxing agent, a repairing solution and an endogenous peroxidase blocking agent.
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| US20170122850A1 (en) * | 2015-11-04 | 2017-05-04 | Marc Key | Method of pretreatment of biological samples for an analyte-staining assay method |
| CN109827821A (en) * | 2019-03-14 | 2019-05-31 | 武汉原谷生物科技有限责任公司 | A kind of non-dimethylbenzene dewaxing renovation agent and preparation method thereof for paraffin section |
| CN113686634A (en) * | 2021-08-10 | 2021-11-23 | 杭州迪英加科技有限公司 | Dewaxing repair liquid beneficial to acquisition of paraffin section images |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170122850A1 (en) * | 2015-11-04 | 2017-05-04 | Marc Key | Method of pretreatment of biological samples for an analyte-staining assay method |
| CN109827821A (en) * | 2019-03-14 | 2019-05-31 | 武汉原谷生物科技有限责任公司 | A kind of non-dimethylbenzene dewaxing renovation agent and preparation method thereof for paraffin section |
| CN113686634A (en) * | 2021-08-10 | 2021-11-23 | 杭州迪英加科技有限公司 | Dewaxing repair liquid beneficial to acquisition of paraffin section images |
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