CN111006928B - Sealing liquid applied to immunohistochemistry and application method thereof - Google Patents
Sealing liquid applied to immunohistochemistry and application method thereof Download PDFInfo
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- CN111006928B CN111006928B CN201911364741.7A CN201911364741A CN111006928B CN 111006928 B CN111006928 B CN 111006928B CN 201911364741 A CN201911364741 A CN 201911364741A CN 111006928 B CN111006928 B CN 111006928B
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- 238000003364 immunohistochemistry Methods 0.000 title claims abstract description 26
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- 108010031186 Glycoside Hydrolases Proteins 0.000 claims abstract description 21
- 230000000694 effects Effects 0.000 claims abstract description 20
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 19
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 19
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 claims abstract description 18
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 claims abstract description 18
- 239000000427 antigen Substances 0.000 claims abstract description 13
- 102000036639 antigens Human genes 0.000 claims abstract description 13
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
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Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a sealing liquid applied to immunohistochemistry and a use method thereof, wherein the sealing liquid comprises the following components in parts by weight: 1000mL of 1 XTBS (Tris-hydrochloric acid buffer), 20-60g of Bovine Serum Albumin (BSA) and 14000U/mL-28000U/mL of glycosidase (PNGase F), and the invention relates to the technical field of clinical medical pathology detection. Compared with the common immunohistochemical sealing liquid, the sealing liquid has the advantages that glycosidase (PNGase F) is added into the sealing liquid, so that N-linked glycosyl of protein can be cut off by the sealing liquid, an antigenic determinant is exposed, a final chromogenic signal is enhanced, and aiming at an antigen containing an N-linked glycosylation modification site, the sealing liquid has better effect, the chromogenic signal of the antigen in immunohistochemical detection is greatly enhanced, the sealing liquid is simple to prepare and low in cost, and the detection step of the immunohistochemistry is not additionally increased.
Description
Technical Field
The invention relates to the technical field of clinical medicine pathology detection, in particular to a sealing liquid applied to immunohistochemistry and a use method thereof.
Background
Immunohistochemistry is a detection technique for qualitative, localized and quantitative determination of a target antigen. Immunohistochemistry combines the specificity of immune response with the visibility of histochemistry, and detects various antigenic substances such as proteins, polypeptides, enzymes, hormones, pathogens, receptors, etc. at the tissue, cell, subcellular level by means of imaging and magnification of a microscope such as a conventional microscope, electron microscope, etc.
In an immunohistochemical reaction, antibodies are able to recognize an epitope and then bind specifically thereto. The blocking solution in the immunohistochemical detection process mainly has the action principle that proteins (such as bovine serum albumin, casein and the like) in the blocking solution are non-specifically combined with non-specific antigenic determinants on the surface of a tissue slice, so that specific antibodies cannot be combined with the non-specific antigenic determinants, and the background signal of color development is reduced. The target epitope is very specific to the antibody, has strong binding capacity and can still be bound after blocking. However, if the blocking time of the blocking solution is too long or the protein concentration is too high, specific binding of the antibody is also affected, resulting in weakening of the final color signal. The blocking solution used in immunohistochemistry comprises animal serum, skimmed milk powder, casein, etc.
In addition, some antigens such as PD-L1, B7-H3, CD7, D22, CD31, CD133, etc. have many glycosylation modifications, and the sugar chain can mask its antigenic determinant, which results in a reduced or no recognition of the antigenic determinant by the specific antibody, making it difficult to accurately reflect the expression of the target in the tissue or cell. After treatment with glycosidase (PNGase F), the N-linked sugar chains are cut off and the epitope is exposed, and specific antibodies recognize and bind to the corresponding epitope, thereby increasing the sensitivity and accuracy of the antibodies to the detection of the target in the tissue or cell.
The glycosidase (PNGase F) added in the sealing liquid has good deglycosylation effect on antigens containing N-linked glycosylation modification, can obviously increase the signal intensity of color development, does not additionally increase the operation steps of immunohistochemical detection, has simple preparation method and low cost, and can be stored for at least two weeks at the temperature of 4 ℃.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the invention provides a blocking solution applied to immunohistochemistry and a use method thereof, and solves the problems that the existing blocking solution has more glycosylation modification, and sugar chains can mask antigenic determinants, so that the recognition capability of specific antibodies on the antigenic determinants is weakened or can not be recognized, and the expression condition of the target in tissues or cells is difficult to accurately reflect.
(II) technical scheme
In order to achieve the above purpose, the invention is realized by the following technical scheme: the sealing liquid for immunohistochemistry comprises the following components in parts by weight: 1000mL of 1 XTBS (Tris-HCl buffer), 20-60g of Bovine Serum Albumin (BSA) and 14000U/mL-28000U/mL of glycosidase (PNGase F).
Preferably, the pH of the 1 XTBS (Tris-HCl buffer) is 8.0.
Preferably, the blocking solution comprises glycosidase (PNGase F) enzyme at a concentration of at least 14000U/mL activity.
Preferably, the preparation method specifically comprises the following steps:
s1, firstly, 100mL of 1 XTBS (Tris-base buffer solution) with the pH of 8.0 is measured and placed in a beaker;
s2, weighing the formula amount of Bovine Serum Albumin (BSA), adding the formula amount of BSA into the solution, and stirring to completely dissolve the BSA;
and S3, adding the glycosidase (PNGase F) with the formula amount into the solution obtained in the step S2, and stirring to completely dissolve the glycosidase, thereby preparing the sealing liquid applied to immunohistochemistry.
The invention also discloses a using method of the sealing liquid applied to immunohistochemistry, which comprises dewaxing treatment, antigen restoration and immunohistochemical staining of paraffin tissue sections, wherein the dewaxing treatment method of the paraffin tissue sections specifically comprises the following steps:
t1, inserting the tissue slice into a slice frame, and soaking the tissue slice in xylene (I) and xylene (II) for 20min respectively;
t2, respectively soaking in absolute ethyl alcohol (I), absolute ethyl alcohol (II), 95% ethyl alcohol, 80% ethyl alcohol and 60% ethyl alcohol for 5min;
t3, soaking and washing in deionized water for three times for 1min each time, and finally transferring into TBS buffer solution with pH of 8.0 for later use;
the antigen repairing method comprises the following specific steps: taking about 500ml of Tris-EDTA repairing liquid in a repairing container, at the moment, ensuring that the repairing liquid can permeate a tissue slice, preheating the repairing liquid to boiling by a microwave oven, then placing the slice into the repairing container, continuously heating the repairing liquid for 15min, maintaining the temperature at 90-100 ℃, and naturally cooling for 30-40min to room temperature after the continuous heating is finished;
the immunohistochemical staining method specifically comprises the following steps:
e1, soaking, washing and cooling the tissue slices with a buffer solution for three times, 1min each time, and then transferring the tissue slices into a TBS buffer solution with the pH value of 8.0 for later use;
e2, taking out the tissue slice in the step E1, wiping the redundant liquid by using absorbent paper, and dripping a proper amount of 3% H on the tissue slice 2 O 2 Incubating and inactivating the aqueous solution in an incubation wet box for 10min at room temperature;
e3, soaking and washing the tissue slices for three times by using TBS buffer solution with pH of 8.0 for 1min each time, then wiping off redundant liquid by using water absorption paper, dripping a proper amount of sealing liquid, and incubating in an incubation wet box for 30min at 37 ℃;
e4, wiping excessive liquid by using water absorption paper, dripping a proper amount of primary antibody reagent, and incubating in a wet box for 1.5h at room temperature;
e5, soaking and washing the tissue slices for three times by using TBS buffer solution with pH of 8.0, wiping excessive liquid by using water absorption paper for 1min each time, dripping a proper amount of HRP-marked goat anti-mouse secondary antibody reagent, and incubating for 30min at room temperature in a wet box;
e6, soaking and washing the tissue slices for three times by using TBS buffer solution with the pH value of 8.0, wiping off redundant liquid by using water absorption paper for 1min each time, dripping a proper amount of prepared DAB color development liquid, timely washing the redundant DAB color development liquid by using deionized water after reacting for 1-5min, and placing the tissue slices into TBS buffer solution with the pH value of 8.0 for later use;
e7, wiping excessive liquid by using water-absorbing paper, dripping a proper amount of hematoxylin staining solution, staining for 2min, washing with TBS buffer solution with pH of 8.0, and transferring into a slicing frame to be soaked with TBS buffer solution with pH of 8.0 for reverse blue 5min;
e8, soaking and washing for three times by deionized water, sequentially adding into 60% ethanol, 80% ethanol, 95% ethanol, absolute ethanol (III), absolute ethanol (IV), dimethylbenzene (III) and dimethylbenzene (IV) to be soaked for 5min respectively, and finally taking out and airing for 5min by a fume hood;
e9, sealing the neutral resin, and observing the dyeing result under a microscope.
Preferably, the use method is to use the sealing liquid disclosed by the invention to replace the conventional sealing liquid.
Preferably, the sealing liquid has the best effect when being prepared and used at present, and can be stored for at least two weeks under the refrigerating condition of 4 ℃.
Preferably, when the sealing liquid is used, the sealing time is 15min-24h, and the sealing temperature is 4-40 ℃.
Preferably, when the sealing liquid is used, the optimal sealing conditions are as follows: sealing at 37deg.C for 30min.
According to experimental analysis, when the concentration of glycosidase (PNGase F) in a formula of the sealing liquid is 14000-28000U/mL, the sealing effect is optimal, when the concentration of glycosidase (PNGase F) is lower than 14000U/mL, the enzyme digestion effect is not obvious and can not reach a better enzyme digestion effect by prolonging the enzyme digestion time, the final coloring effect is poor, when the concentration of glycosidase (PNGase F) is higher than 28000U/mL, the final coloring is not obviously enhanced, the consumption of glycosidase (PNGase F) is more wasteful, and when the sealing time is 15 minutes-24 hours, the enzyme digestion effect is best, and the sealing effect can be ensured; when the sealing temperature is 4-40 ℃, the enzyme digestion effect on glycosylation groups is best, and the sealing effect is not influenced.
(III) beneficial effects
The invention provides a sealing liquid applied to immunohistochemistry and a using method thereof. Compared with the prior art, the method has the following beneficial effects:
(1) Compared with the common immunohistochemical sealing liquid, the sealing liquid has the advantages that glycosidase (PNGase F) is added into the sealing liquid, so that N-linked glycosyl of protein can be cut off by the sealing liquid, an antigenic determinant is exposed, and a final chromogenic signal is enhanced.
(2) The blocking solution applied to immunohistochemistry and the application method thereof have better effect aiming at antigens containing N-linked glycosylation modification sites, and greatly enhance the color development signals of the antigens in immunohistochemical detection.
(3) The sealing liquid applied to immunohistochemistry and the application method thereof have the advantages of simple preparation, low cost and no additional detection step of the immunohistochemistry.
Drawings
FIG. 1 is a graph showing the effect of the invention in the dyeing results of example 1, showing the result of the dyeing of the experimental group of chronic tonsillitis;
FIG. 2 is a graph showing the result of dyeing in example 1 of the effect of the present invention, showing the result of dyeing in the control group for chronic tonsillitis;
FIG. 3 is a chart showing the result of dyeing of experimental groups of placenta among the results of dyeing in example 2 of the effect of the present invention;
FIG. 4 is a chart showing the result of staining of the placenta control group in example 2;
FIG. 5 is a chart showing the staining of experimental groups for breast cancer in the staining results of example 2, which is an effect of the present invention;
FIG. 6 is a graph showing the result of staining of the breast cancer control group in example 2.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-6, two technical schemes are provided in the embodiment of the present invention: a blocking solution applied to immunohistochemistry and a using method thereof are provided, wherein the following examples adopt the following reagent specifications and purities:
1 XTBS (Tris-HCl buffer) at pH 8.0:
sodium chloride (NaCl), analytical grade, cat No. 10019318, national pharmaceutical chemicals limited.
Tris (Tris-base), ultrapure, cat# 3077C308-5KG,VWR International, LLC.
Hydrochloric acid, analytical grade, cat No. 10011018, national drug group chemical company, inc.
Double distilled water (ddH) 2 O), double distilled water, self-prepared by this company.
Bovine Serum Albumin (BSA), cat No. 1257c319,Amresco product.
Glycosidase (PNGase F), 1,800,000U/mg,0.4mg/mL, ATE00005 (stock number ATA 808), general Biotechnology Co., ltd (AtaGenix).
The preparation method of the sealing liquid applied to immunohistochemistry specifically comprises the following examples:
example 1
100mL of 1 XTBS (Tris-base buffer) with pH of 8.0 is measured and placed in a beaker; 3g of Bovine Serum Albumin (BSA) was weighed and added to the above solution, and stirred to be completely dissolved; 1.95mL of glycosidase (PNGase F) was added to the above solution and stirred to dissolve completely. Temporary storage is carried out at the temperature of 4 ℃ for standby.
Example 2
100mL of 1 XTBS (Tris-base buffer) at pH8.0 was measured in a beaker; 3g of Bovine Serum Albumin (BSA) was weighed and added to the above solution, and stirred to be completely dissolved; to the above solution, 3.89mL of glycosidase (PNGase F) was added and stirred to dissolve completely. Temporary storage is carried out at the temperature of 4 ℃ for standby.
A method for using a blocking solution applied to immunohistochemistry, which specifically comprises the following embodiments:
effect example 1
After the preparation of the sealing liquid according to example 1 was completed, two chronic tonsillitis tissue sections of the same origin were respectively subjected to immunohistochemical PD-L1 staining according to the standard experimental procedure of the present invention, one for the experimental group, and sealed with the sealing liquid of the present invention; one is a control group, the blocking solution used in the control group does not contain glycosidase (PNGase F), and the other components are completely consistent with the experimental group.
The method comprises the following specific steps:
the paraffin tissue section dewaxing treatment method specifically comprises the following steps:
t1, inserting the tissue slice into a slice frame, and soaking in the xylene (I) and the xylene (II) for 20min respectively.
T2, soaking in absolute ethyl alcohol (I), absolute ethyl alcohol (II), 95% ethyl alcohol, 80% ethyl alcohol and 60% ethyl alcohol for 5min respectively.
And T3, soaking and washing in deionized water for three times, each time for 1min, and finally transferring into TBS buffer solution with pH of 8.0 for later use.
The antigen repairing method comprises the following specific steps:
taking about 500ml of Tris-EDTA repairing liquid in a repairing container, preheating the repairing liquid to boiling by a microwave oven to ensure that the repairing liquid can permeate through a tissue slice, then placing the slice into the repairing container, continuously heating the repairing liquid for 15min, maintaining the temperature at 95 ℃, naturally cooling to room temperature after the continuous heating is finished, and cooling to room temperature after 30-40min is usually performed.
The immunohistochemical staining method specifically comprises the following steps:
e1, soaking, washing and cooling the tissue slices three times by using TBS buffer solution with the pH value of 8.0, 1min each time, and then transferring the tissue slices into TBS buffer solution with the pH value of 8.0 for later use.
E2, taking out the tissue slice in the step E1, wiping the redundant liquid by using absorbent paper, and dripping a proper amount of 3% H on the tissue slice 2 O 2 The aqueous solution was incubated in an incubation wet box for 10min at room temperature for inactivation.
And E3, soaking and washing the tissue slices three times by using TBS buffer solution with pH of 8.0 for 1min each time, then wiping off redundant liquid by using water absorption paper, dripping a proper amount of sealing liquid according to the invention, and incubating in an incubation wet box for 30min at 37 ℃.
And E4, wiping excessive liquid by using water absorption paper, dripping a proper amount of PD-L1 primary antibody reagent, and incubating in a wet box for 1.5h at room temperature.
E5, soaking and washing the tissue slices for three times by using TBS buffer solution with pH of 8.0, wiping excessive liquid by using water absorption paper for 1min each time, dripping a proper amount of HRP-marked goat anti-mouse secondary antibody reagent, and incubating for 30min at room temperature in a wet box.
E6, soaking and washing the tissue slices for three times by using TBS buffer solution with the pH value of 8.0, wiping the redundant liquid by using water absorbing paper for 1min each time, dripping a proper amount of prepared DAB color development liquid, timely washing the redundant DAB color development liquid by using deionized water after reacting for 1-5min, and placing the tissue slices into the TBS buffer solution with the pH value of 8.0 for later use.
And E7, wiping excessive liquid by using water-absorbing paper, dripping a proper amount of hematoxylin staining solution, staining for 2min, washing with TBS buffer solution with pH of 8.0, and transferring into a slicing frame to be soaked with TBS buffer solution with pH of 8.0 for reverse blue 5min.
E8, soaking and washing for three times by deionized water, sequentially adding into 60% ethanol, 80% ethanol, 95% ethanol, absolute ethanol (III), absolute ethanol (IV), dimethylbenzene (III) and dimethylbenzene (IV) to soak for 5min respectively, and finally taking out and airing for 5min by a fume hood.
E7, sealing the neutral resin, and observing the dyeing result under a microscope.
The experimental results are explained as follows:
by comparing the pictures of the experimental group and the control group, the target position is more deeply colored after the color development by using the sealing liquid provided by the invention, and the positive result is obvious. The expression condition of PD-L1 in the tissue slice can be accurately judged.
Effect example 2
After the preparation of the blocking solution according to example 2, immunohistochemical PD-L1 staining was performed on two placental tissue sections of the same origin and two breast cancer tissue sections of the same origin, respectively, according to the standard experimental procedure of the present invention. One of the two different tissue sections is an experimental group, and the sealing liquid is used for sealing; the other control group was used without glycosidase (PNGase F) in the blocking solution, and the other components were identical to the experimental group.
The method comprises the following specific steps:
the paraffin tissue section dewaxing treatment method specifically comprises the following steps:
t1, inserting the tissue slice into a slice frame, and soaking in the xylene (I) and the xylene (II) for 20min respectively.
T2, soaking in absolute ethyl alcohol (I), absolute ethyl alcohol (II), 95% ethyl alcohol, 80% ethyl alcohol and 60% ethyl alcohol for 5min respectively.
And T3, soaking and washing in deionized water for three times, each time for 1min, and finally transferring into TBS buffer solution with pH of 8.0 for later use.
The antigen repairing method comprises the following specific steps:
taking about 500ml of Tris-EDTA repairing liquid in a repairing container, preheating the repairing liquid to boiling by a microwave oven to ensure that the repairing liquid can permeate through a tissue slice, then placing the slice into the repairing container, continuously heating the repairing liquid for 15min, maintaining the temperature at 95 ℃, naturally cooling to room temperature after the continuous heating is finished, and cooling to room temperature after 30-40min is usually performed.
The immunohistochemical staining method specifically comprises the following steps:
e1, soaking, washing and cooling the tissue slices three times by using TBS buffer solution with the pH value of 8.0, 1min each time, and then transferring the tissue slices into TBS buffer solution with the pH value of 8.0 for later use.
E2, taking out the tissue slice, wiping the redundant liquid by using water absorbing paper, and dripping a proper amount of 3%H on the tissue slice 2 O 2 The aqueous solution was incubated in an incubation wet box for 10min at room temperature for inactivation.
E3, soaking and washing the tissue slices for three times by using TBS buffer solution with the pH value of 8.0, wiping off redundant liquid by using water absorption paper for 1min each time, dripping a proper amount of the sealing liquid of the invention into an experimental group, dripping the sealing liquid of the same amount of a control group into a control group, and incubating for 30min in an incubation wet box at 37 ℃.
And E4, wiping excessive liquid by using water absorption paper, dripping a proper amount of PD-L1 primary antibody reagent, and incubating in a wet box for 1.5h at room temperature.
E5, soaking and washing the tissue slices for three times with TBS buffer solution with pH of 8.0, wiping off redundant liquid with absorbent paper for 1min each time, dripping a proper amount of HRP-marked goat anti-mouse secondary antibody reagent, and incubating for 30min at room temperature in a wet box.
E6, soaking and washing the tissue slices for three times by using TBS buffer solution with the pH value of 8.0, wiping the redundant liquid by using water absorbing paper for 1min each time, dripping a proper amount of prepared DAB color development liquid, timely washing the redundant DAB color development liquid by using deionized water after reacting for 1-5min, and placing the tissue slices into the TBS buffer solution with the pH value of 8.0 for later use.
And E7, wiping excessive liquid by using water absorbing paper, dripping a proper amount of hematoxylin staining solution, staining for 2min, washing with TBS buffer solution with pH of 8.0, and transferring into a slicing frame to be soaked with TBS buffer solution with pH of 8.0 for reverse blue 5min.
E8, soaking and washing for three times by deionized water solution, sequentially adding into 60% ethanol, 80% ethanol, 95% ethanol, absolute ethanol (III), absolute ethanol (IV), dimethylbenzene (III) and dimethylbenzene (IV) to soak for 5min respectively, and finally taking out and airing for 5min by a fume hood.
E9, sealing the neutral resin, and observing the dyeing result under a microscope.
The experimental results are explained as follows:
by comparing the experimental group and the control group of the placenta tissue slice with the experimental group and the control group of the breast cancer tissue slice, the invention obviously shows that the sealing liquid can lead the tissue slice to have stronger coloring signals after being dyed, and the PD-L1 expression condition in the tissue slice can be more accurately judged by using the sealing liquid through the comparison.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A blocking solution for immunohistochemistry, characterized in that: the sealing liquid comprises the following components in parts by weight: 1000mL of 1 XTBS, 20-60g of bovine serum albumin and a glycosidase PNGase F with an activity of 14000-28000U/mL.
2. A blocking fluid for use in immunohistochemistry according to claim 1, wherein: the pH of the 1 XTBS was 8.0.
3. A blocking fluid for use in immunohistochemistry according to claim 1, wherein: the preparation method specifically comprises the following steps:
s1, firstly, 100mL of 1 XTBS with the pH of 8.0 is measured and placed in a beaker;
s2, weighing the bovine serum albumin with the formula amount, adding the bovine serum albumin into the solution obtained in the S1, and stirring to completely dissolve the bovine serum albumin;
and S3, adding the glycosidase with the formula amount into the solution obtained in the step S2, and stirring to completely dissolve the glycosidase, thereby preparing the sealing liquid applied to immunohistochemistry.
4. A method for using a blocking solution applied to immunohistochemistry, which is characterized in that: the method comprises dewaxing treatment, antigen retrieval and immunohistochemical staining of paraffin tissue sections, wherein the dewaxing treatment method of the paraffin tissue sections specifically comprises the following steps:
t1, inserting the tissue slice into a slice frame, and soaking the tissue slice in xylene I and xylene II for 20min respectively;
t2, respectively soaking in absolute ethyl alcohol I, absolute ethyl alcohol II, 95% ethyl alcohol, 80% ethyl alcohol and 60% ethyl alcohol for 5min;
t3, soaking and washing in deionized water for three times for 1min each time, and finally transferring into TBS buffer solution with pH of 8.0 for later use;
the antigen repairing method comprises the following specific steps: taking about 500ml of Tris-EDTA repairing liquid in a repairing container, at the moment, ensuring that the repairing liquid can permeate a tissue slice, preheating the repairing liquid to boiling by a microwave oven, then placing the slice into the repairing container, continuously heating the repairing liquid for 15min, maintaining the temperature at 90-100 ℃, and naturally cooling for 30-40min to room temperature after the continuous heating is finished;
the immunohistochemical staining method specifically comprises the following steps:
e1, soaking, washing and cooling the tissue slices with a buffer solution for three times, 1min each time, and then transferring the tissue slices into a TBS buffer solution with the pH value of 8.0 for later use;
e2, taking out the tissue slice in the step E1, wiping the redundant liquid by using absorbent paper, and dripping a proper amount of 3% H on the tissue slice 2 O 2 Incubating and inactivating the aqueous solution in an incubation wet box for 10min at room temperature;
e3, soaking and washing the tissue slices three times by using TBS buffer solution with pH of 8.0 for 1min each time, then wiping the redundant liquid by using absorbent paper, dripping a proper amount of sealing liquid according to claim 1, and incubating in an incubation wet box for 30min at 37 ℃;
e4, wiping excessive liquid by using water absorption paper, dripping a proper amount of primary antibody reagent, and incubating in a wet box for 1.5h at room temperature;
e5, soaking and washing the tissue slices for three times by using TBS buffer solution with pH of 8.0, wiping excessive liquid by using water absorption paper for 1min each time, dripping a proper amount of HRP-marked goat anti-mouse secondary antibody reagent, and incubating for 30min at room temperature in a wet box;
e6, soaking and washing the tissue slices for three times by using TBS buffer solution with the pH value of 8.0, wiping off redundant liquid by using water absorption paper for 1min each time, dripping a proper amount of prepared DAB color development liquid, timely washing the redundant DAB color development liquid by using deionized water after reacting for 1-5min, and putting the tissue slices into TBS buffer solution with the pH value of 8.0 for later use;
e7, wiping excessive liquid by using water-absorbing paper, dripping a proper amount of hematoxylin staining solution, staining for 2min, washing with TBS buffer solution with pH of 8.0, and transferring into a slicing frame to be soaked in TBS buffer solution with pH of 8.0 for reverse blue for 5min;
e8, soaking and washing for three times by deionized water, sequentially putting into 60% ethanol, 80% ethanol, 95% ethanol, absolute ethanol III, absolute ethanol IV, xylene III and xylene IV, respectively soaking for 5min, and finally taking out and airing for 5min in a fume hood;
e9, sealing the neutral resin, and observing the dyeing result under a microscope.
5. The method of using a blocking solution for immunohistochemistry according to claim 4, wherein: the use method is to replace the conventional sealing liquid with the sealing liquid in claim 4.
6. The method of using a blocking solution for immunohistochemistry according to claim 4, wherein: the sealing liquid needs to be prepared at present or stored for at least two weeks under the refrigerating condition of 4 ℃.
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| CN113495138A (en) * | 2021-06-22 | 2021-10-12 | 南京弗瑞思生物科技有限公司 | Tissue slice antigen repair liquid and use method thereof |
| CN115541872A (en) * | 2022-09-22 | 2022-12-30 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | PD-L1 detection kit and detection method thereof |
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