CN111006928A - Sealing liquid applied to immunohistochemistry and using method thereof - Google Patents
Sealing liquid applied to immunohistochemistry and using method thereof Download PDFInfo
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- CN111006928A CN111006928A CN201911364741.7A CN201911364741A CN111006928A CN 111006928 A CN111006928 A CN 111006928A CN 201911364741 A CN201911364741 A CN 201911364741A CN 111006928 A CN111006928 A CN 111006928A
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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
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Abstract
The invention discloses a sealing liquid applied to immunohistochemistry and a using method thereof, wherein the sealing liquid comprises the following components in parts by weight: the invention relates to the technical field of clinical medicine pathological detection, in particular to 1000mL of 1 xTBS (Tris hydrochloric acid buffer), 20-60g of Bovine Serum Albumin (BSA) and 14000U/mL-28000U/mL of glycosidase (PNGase F). Compared with the common immunohistochemical sealing liquid, the invention adds glycosidase (PNGase F) into the sealing liquid, so that the N-linked glycosyl of protein can be cut off by the sealing liquid, antigenic determinant is exposed, and further the final chromogenic signal is enhanced.
Description
Technical Field
The invention relates to the technical field of clinical medical pathological detection, in particular to a confining liquid applied to immunohistochemistry and a using method thereof.
Background
Immunohistochemistry is a detection technique for qualitative, localized, and quantitative determination of target antigens. Immunohistochemistry skillfully combines the specificity of immune reaction and histochemical visibility, and detects various antigen substances such as proteins, polypeptides, enzymes, hormones, pathogens, receptors and the like at the level of tissues, cells and subcellular cells by means of the imaging and amplifying effects of microscopes such as ordinary microscopes, electron microscopes and the like.
In immunohistochemistry, antibodies are able to recognize an antigenic determinant, which then binds specifically thereto. The action principle of the blocking solution in the immunohistochemical detection process is mainly 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 section, so that specific antibodies cannot be combined with the non-specific antigenic determinants, and the background signal of color development is reduced. And the target antigenic determinant is combined with the antibody very specifically, has strong combining capacity and can still be combined after being blocked. However, if the blocking time of the blocking solution is too long or the protein concentration is too high, the specific binding of the antibody is also affected, resulting in a weakening of the final chromogenic signal. Common blocking solutions for immunohistochemistry include animal serum, skimmed milk powder, casein, etc.
In addition, some antigens such as PD-L1, B7-H3, CD7, D22, CD31, CD133 and the like have more glycosylation modifications, and the sugar chain can mask the epitope, so that the recognition capability of the specific antibody to the epitope is reduced or can not be recognized, and the expression condition of the target in the tissue or cell is difficult to accurately reflect. After treatment with glycosidase (PNGase F), the N-linked sugar chains are cleaved, the epitopes are exposed, and the specific antibody recognizes and binds the corresponding epitope, thereby increasing the sensitivity and accuracy of the antibody for detection of the target in a tissue or cell.
The glycosidase (PNGase F) added in the sealing liquid has good deglycosylation effect on the antigen 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 4 ℃.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a sealing liquid applied to immunohistochemistry and a using method thereof, and solves the problems that the existing sealing liquid has more glycosylation modifications, and the carbohydrate chain can shield the antigenic determinant thereof, so that the recognition capability of a specific antibody to the antigenic determinant is weakened or the specific antibody cannot be recognized, and the expression condition of the target in the tissue or the cell is difficult to accurately reflect.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a sealing liquid applied to immunohistochemistry comprises the following components in parts by weight: 1000mL of 1 XBS (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 × TBS (Tris-HCl buffer) is 8.0.
Preferably, the blocking solution comprises a glycosidase (PNGase F) enzyme in an active concentration of at least 14000U/mL.
Preferably, the preparation method specifically comprises the following steps:
s1, firstly, measuring 100mL of 1 × TBS (Tris-base buffer solution) with the pH value of 8.0 in a beaker;
s2, weighing Bovine Serum Albumin (BSA) with the formula amount, adding the Bovine Serum Albumin (BSA) into the solution, and stirring to completely dissolve the BSA;
s3, adding 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 blocking solution applied to immunohistochemistry.
The invention also discloses a using method of the sealing liquid applied to immunohistochemistry, which comprises the steps of dewaxing treatment of paraffin tissue sections, antigen retrieval and immunohistochemical staining, wherein the dewaxing treatment method of the paraffin tissue sections specifically comprises the following steps:
t1, inserting the tissue slices into a slice rack, and soaking in xylene (I) and xylene (II) for 20min respectively;
t2, respectively soaking in anhydrous ethanol (I), anhydrous ethanol (II), 95% ethanol, 80% ethanol and 60% ethanol for 5 min;
t3, soaking and washing the mixture in deionized water for three times, wherein each time is 1min, and finally transferring the mixture into TBS buffer solution with the pH value of 8.0 for later use;
the antigen retrieval method comprises the following specific steps: taking about 500ml of Tris-EDTA repair liquid into a repair container, ensuring that the repair liquid can submerge tissue slices, preheating the repair liquid by using a microwave oven until the tissue slices are boiled, then putting the slices into the repair container, continuously heating the repair liquid for 15min, keeping the temperature at 90-100 ℃, and naturally cooling for 30-40min to room temperature after the continuous heating is finished;
the method for immunohistochemical staining specifically comprises the following steps:
e1, soaking and washing the cooled tissue slices for three times by using buffer solution, wherein each time is 1min, 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 off redundant liquid by using absorbent paper, and dripping a proper amount of 3% H on the tissue slice2O2Incubating 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, 1min each time, wiping off redundant liquid by using absorbent paper, dropwise adding a proper amount of the confining liquid, and incubating in an incubation wet box for 30min at 37 ℃;
e4, wiping off redundant liquid by using absorbent paper, dripping a proper amount of primary antibody reagent, and incubating in a wet box for 1.5h at room temperature;
e5, washing the tissue slice three times with TBS buffer solution with pH8.0, 1min each time, wiping off the redundant liquid with absorbent paper, dripping appropriate amount of HRP-labeled goat-anti-mouse secondary antibody reagent, and incubating in a wet box at room temperature for 30 min;
e6, soaking and washing the tissue slices for three times by TBS buffer solution with pH of 8.0, 1min each time, wiping off redundant liquid by absorbent paper, dripping a proper amount of prepared DAB color developing solution, washing the redundant DAB color developing solution by deionized water in time after reacting for 1-5min, and placing the tissue slices in the TBS buffer solution with pH of 8.0 for standby;
e7, wiping off redundant liquid by absorbent paper, dripping a proper amount of hematoxylin staining solution, staining for 2min, washing with TBS buffer solution with pH of 8.0, transferring to a slicing frame, and soaking in TBS buffer solution with pH of 8.0 for 5 min;
e8, soaking and washing with deionized water for three times, sequentially soaking in 60% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol (III), anhydrous ethanol (IV), xylene (III) and xylene (IV) for 5min, and air drying in a fume hood for 5 min;
e9, sealing with neutral gum, and observing the dyeing result under a microscope.
Preferably, the use method is to use the sealing liquid of the invention to replace the conventional sealing liquid.
Preferably, the confining liquid is best when it is prepared as it is, and can also be stored under refrigeration at 4 ℃ for at least two weeks.
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: blocking at 37 ℃ for 30 min.
The experimental analysis shows that the sealing effect is optimal when the concentration of glycosidase (PNGase F) in the formula of the sealing liquid is 14000-; when the blocking temperature is 4-40 ℃, the enzyme digestion effect on the glycosylation group is the best, and the blocking effect is not influenced.
(III) advantageous effects
The invention provides a confining 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 invention adds glycosidase (PNGase F) into the sealing liquid, so that the N-linked glycosyl of the protein can be cut off by the sealing liquid, antigenic determinants are exposed, and the final chromogenic signal is further enhanced.
(2) The sealing liquid applied to immunohistochemistry and the use method thereof have better effect aiming at the antigen containing the N-linked glycosylation modification sites, and greatly enhance the chromogenic signal of the antigen in immunohistochemical detection.
(3) The sealing liquid applied to immunohistochemistry and the use method thereof have the advantages of simple preparation of the sealing liquid, low cost and no additional detection step of immunohistochemistry.
Drawings
FIG. 1 is a graph showing staining patterns of a chronic tonsillitis test group in staining results of example 1 according to the present invention;
FIG. 2 is a graph showing the staining of a control group of chronic tonsillitis in the staining results of example 1 according to the present invention;
FIG. 3 is a dyeing chart of experimental group of placenta in dyeing results of example 2 according to the present invention;
FIG. 4 is a graph showing the staining results of a placenta control group in example 2;
FIG. 5 is a staining pattern of an experimental group of breast cancer in the staining result of example 2 according to the present invention;
FIG. 6 is a staining pattern of a breast cancer control group in the staining result of example 2 according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-6, the embodiment of the present invention provides two technical solutions: the sealing liquid applied to immunohistochemistry and the use method thereof adopt the following reagent specifications and purities in the following embodiments:
1 × TBS (Tris HCl buffer) at pH8.0 contains the following components:
sodium chloride (NaCl), analytical grade, cat # 10019318, national drug group chemical reagents ltd.
Tris-base, ultrapure, cat 3077C308-5KG, VWR International, LLC.
Hydrochloric acid, analytical grade, cat # 10011018, national pharmaceutical group chemical reagents ltd.
Double distilled water (ddH)2O), double distilled water, self-prepared by this company.
Bovine Serum Albumin (BSA), cat # 1257c319, Amresco product.
Glycosidase (PNGase F), 1,800,000U/mg, 0.4mg/mL, Cat # ATE00005 (Cat # ATA 808), Probiota (Wuhan) science and technology Co., Ltd. (AtaGenix).
A preparation method of a sealing liquid applied to immunohistochemistry specifically comprises the following embodiments:
example 1
Measuring 100mL of 1 × TBS (Tris-base buffer solution) with pH of 8.0 in a beaker; weighing 3g of Bovine Serum Albumin (BSA) and adding the BSA into the solution, and stirring to completely dissolve the BSA; 1.95mL of glycosidase (PNGase F) was added to the above solution, and the mixture was stirred to dissolve completely. Temporarily storing at 4 ℃ for later use.
Example 2
Measuring 100mL of 1 × TBS (Tris-base buffer solution) with pH of 8.0 in a beaker; weighing 3g of Bovine Serum Albumin (BSA) and adding the BSA into the solution, and stirring to completely dissolve the BSA; 3.89mL of glycosidase (PNGase F) was added to the above solution, and the mixture was stirred to dissolve completely. Temporarily storing at 4 ℃ for later use.
The use method of the sealing liquid applied to immunohistochemistry specifically comprises the following embodiments:
effect example 1
After the sealing liquid is prepared according to the example 1, immunohistochemical PD-L1 staining is respectively carried out on two chronic tonsillitis tissue sections with the same source according to the standard experimental process of the invention, and one is an experimental group which is sealed by using the sealing liquid of the 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 identical to those in 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 slices into the slicing frame, and soaking in xylene (I) and xylene (II) for 20min respectively.
T2, soaking in anhydrous ethanol (I), anhydrous ethanol (II), 95% ethanol, 80% ethanol, and 60% ethanol for 5 min.
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 retrieval method comprises the following specific steps:
taking about 500ml of Tris-EDTA repair liquid into a repair container, preheating the repair liquid by using a microwave oven until the repair liquid can submerge tissue slices, then putting the slices into the repair container, continuously heating the repair liquid for 15min, keeping the temperature at 95 ℃, naturally cooling to room temperature after the continuous heating is finished, and cooling to room temperature usually after 30-40 min.
The method for immunohistochemical staining specifically comprises the following steps:
e1, soaking and washing the cooled tissue slices three times by TBS buffer solution with the pH of 8.0, wherein each time is 1min, and then transferring the tissue slices into TBS buffer solution with the pH of 8.0 for standby.
E2, taking out the tissue slice in the step E1, wiping off redundant liquid by using absorbent paper, and dripping a proper amount of 3% H on the tissue slice2O2The aqueous solution was incubated in a wet incubation box at room temperature for 10min for inactivation.
E3, soaking and washing the tissue slices for three times by using TBS buffer solution with pH8.0, 1min each time, then wiping off redundant liquid by using absorbent paper, dropwise adding a proper amount of confining liquid of the invention, and incubating for 30min in an incubation wet box at 37 ℃.
E4, wiping off the redundant liquid by using absorbent paper, dripping a proper amount of PD-L1 primary antibody reagent, and incubating for 1.5h in a wet box at room temperature.
E5, washing tissue slices three times with TBS buffer solution with pH8.0, 1min each time, wiping off excessive liquid with absorbent paper, adding appropriate amount of HRP-labeled goat-anti-mouse secondary antibody reagent dropwise, and incubating in a wet box at room temperature for 30 min.
E6, soaking and washing the tissue section for three times by TBS buffer solution with pH of 8.0, 1min each time, wiping off redundant liquid by absorbent paper, dripping a proper amount of prepared DAB color developing solution, washing the redundant DAB color developing solution by deionized water in time after reacting for 1-5min, and placing the tissue section in TBS buffer solution with pH of 8.0 for standby.
E7, wiping off the redundant liquid by absorbent paper, dripping a proper amount of hematoxylin staining solution, staining for 2min, washing with TBS buffer solution with pH8.0, transferring to a slicing frame, and soaking in TBS buffer solution with pH8.0 for 5 min.
E8, soaking and washing with deionized water for three times, sequentially soaking in 60% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol (III), anhydrous ethanol (IV), xylene (III) and xylene (IV) for 5min, and air drying in a fume hood for 5 min.
E7, sealing with neutral gum, and observing the dyeing result under a microscope.
The experimental results are explained below:
by comparing the pictures of the experimental group and the control group, it is obvious that the target position is more deeply colored after the coloring by using the confining liquid of the invention, and the positive result is obvious. Can more accurately judge the expression condition of PD-L1 in the tissue section.
Effect example 2
After the blocking solution was prepared according to example 2, immunohistochemical PD-L1 staining was performed on two placenta tissue sections from the same source and two breast cancer tissue sections from the same source, respectively, according to the standard experimental procedure of the present invention. One of two different tissue sections is taken as an experimental group, and the sealing liquid is used for sealing; the other panel is a control group, which uses a blocking solution without glycosidase (PNGase F), and the other components are identical to those of 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 slices into the slicing frame, and soaking in xylene (I) and xylene (II) for 20min respectively.
T2, soaking in anhydrous ethanol (I), anhydrous ethanol (II), 95% ethanol, 80% ethanol, and 60% ethanol for 5 min.
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 retrieval method comprises the following specific steps:
taking about 500ml of Tris-EDTA repair liquid into a repair container, preheating the repair liquid by using a microwave oven until the repair liquid can submerge tissue slices, then putting the slices into the repair container, continuously heating the repair liquid for 15min, keeping the temperature at 95 ℃, naturally cooling to room temperature after the continuous heating is finished, and cooling to room temperature usually after 30-40 min.
The method for immunohistochemical staining specifically comprises the following steps:
e1, soaking and washing the cooled tissue section three times by TBS buffer solution with pH of 8.0, each time for 1min, and then transferring the tissue section into TBS buffer solution with pH of 8.0 for standby.
E2, taking out the tissue slice, wiping the redundant liquid with absorbent paper, and dripping a proper amount of over 3% H on the tissue slice2O2The aqueous solution was incubated in a wet incubation box at room temperature for 10min for inactivation.
E3, soaking and washing the tissue section for three times by TBS buffer solution with pH of 8.0, 1min each time, wiping off redundant liquid by absorbent paper, dropwise adding a proper amount of the confining liquid of the invention into the experimental group, dropwise adding the same amount of the confining liquid of the control group into the control group, and incubating for 30min in an incubation wet box at 37 ℃.
E4, wiping off the redundant liquid by using absorbent paper, dripping a proper amount of PD-L1 primary antibody reagent, and incubating for 1.5h in a wet box at room temperature.
E5, and washing the tissue section three times in TBS buffer solution with pH of 8.0, each time for 1min, wiping off the redundant liquid with absorbent paper, dripping a proper amount of HRP-labeled goat-anti-mouse secondary antibody reagent, and incubating in a wet box at room temperature for 30 min.
E6, soaking and washing the tissue slices for three times by TBS buffer solution with the pH of 8.0, 1min each time, wiping off redundant liquid by using absorbent paper, dripping a proper amount of prepared DAB color developing solution, washing the redundant DAB color developing solution by using deionized water in time after reacting for 1-5min, and placing the tissue slices in the TBS buffer solution with the pH of 8.0 for later use.
E7, wiping off the redundant liquid by absorbent paper, dripping a proper amount of hematoxylin staining solution, staining for 2min, washing with TBS buffer solution with pH of 8.0, transferring to a slicing frame, and soaking in TBS buffer solution with pH of 8.0 for 5 min.
E8, soaking and washing with deionized water for three times, sequentially soaking in 60% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol (III), anhydrous ethanol (IV), xylene (III) and xylene (IV) for 5min, and air drying in a fume hood for 5 min.
E9, sealing with neutral gum, and observing the dyeing result under a microscope.
The experimental results are explained below:
by comparing the experimental group and the control group of the placenta tissue section and the experimental group and the control group of the breast cancer tissue section, it is obvious that the confining liquid provided by the invention can enable the tissue section to have stronger coloring signals after being dyed, and by the comparison, the confining liquid provided by the invention can more accurately judge the expression condition of PD-L1 in the tissue section.
It is noted that, herein, relational terms such as first and second, and the like may be 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. Also, 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 appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A confining liquid applied to immunohistochemistry is characterized in that: the sealing liquid comprises the following components in parts by weight: 1000mL of 1 XTSS (Tris-HCl buffer), 20-60g of Bovine Serum Albumin (BSA), and 14000U/mL-28000U/mL of glycosidase (PNGase F).
2. The sealant solution for immunohistochemistry according to claim 1, wherein: the pH of the 1 XTSS (Tris-HCl buffer) was 8.0.
3. The sealant solution for immunohistochemistry according to claim 1, wherein: the preparation method specifically comprises the following steps:
s1, firstly, measuring 100mL of 1 × TBS (Tris-base buffer solution) with the pH value of 8.0 in a beaker;
s2, weighing Bovine Serum Albumin (BSA) with the formula amount, adding the Bovine Serum Albumin (BSA) into the solution, and stirring to completely dissolve the BSA;
s3, adding 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 blocking solution applied to immunohistochemistry.
4. A method for using a sealing liquid applied to immunohistochemistry is characterized in that: the paraffin tissue section dewaxing treatment method comprises the steps of dewaxing treatment of a paraffin tissue section, antigen retrieval and immunohistochemical staining, and specifically comprises the following steps:
t1, inserting the tissue slices into a slice rack, and soaking in xylene (I) and xylene (II) for 20min respectively;
t2, respectively soaking in anhydrous ethanol (I), anhydrous ethanol (II), 95% ethanol, 80% ethanol and 60% ethanol for 5 min;
t3, soaking and washing the mixture in deionized water for three times, wherein each time is 1min, and finally transferring the mixture into TBS buffer solution with the pH value of 8.0 for later use;
the antigen retrieval method comprises the following specific steps: taking about 500ml of Tris-EDTA repair liquid into a repair container, ensuring that the repair liquid can submerge tissue slices, preheating the repair liquid by using a microwave oven until the tissue slices are boiled, then putting the slices into the repair container, continuously heating the repair liquid for 15min, keeping the temperature at 90-100 ℃, and naturally cooling for 30-40min to room temperature after the continuous heating is finished;
the method for immunohistochemical staining specifically comprises the following steps:
e1, soaking and washing the cooled tissue slices for three times by using buffer solution, wherein each time is 1min, 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 off redundant liquid by using absorbent paper, and dripping a proper amount of 3% H on the tissue slice2O2Incubating 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, 1min each time, wiping off redundant liquid by using absorbent paper, dropwise adding a proper amount of the confining liquid, and incubating in an incubation wet box for 30min at 37 ℃;
e4, wiping off redundant liquid by using absorbent paper, dripping a proper amount of primary antibody reagent, and incubating in a wet box for 1.5h at room temperature;
e5, washing the tissue slice three times with TBS buffer solution with pH8.0, 1min each time, wiping off the redundant liquid with absorbent paper, dripping appropriate amount of HRP-labeled goat-anti-mouse secondary antibody reagent, and incubating in a wet box at room temperature for 30 min;
e6, soaking and washing the tissue slices for three times by TBS buffer solution with pH of 8.0, 1min each time, wiping off redundant liquid by using absorbent paper, dropwise adding a proper amount of prepared DAB color developing solution, timely washing the redundant DAB color developing solution by using deionized water after reacting for 1-5min, and placing the tissue slices in the TBS buffer solution with pH of 8.0 for later use;
e7, wiping off redundant liquid by absorbent paper, dripping a proper amount of hematoxylin staining solution, staining for 2min, washing with TBS buffer solution with pH of 8.0, transferring to a slicing frame, and soaking in TBS buffer solution with pH of 8.0 for 5 min;
e8, soaking and washing with deionized water for three times, sequentially soaking in 60% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol (III), anhydrous ethanol (IV), xylene (III) and xylene (IV) for 5min, and air drying in a fume hood for 5 min;
e9, sealing with neutral gum, and observing the dyeing result under a microscope.
5. The method of claim 4, wherein the blocking solution is applied to immunohistochemistry, and the method comprises the following steps: the using method is to use the sealing liquid of the invention to replace the conventional sealing liquid.
6. The method of claim 4, wherein the blocking solution is applied to immunohistochemistry, and the method comprises the following steps: the sealing liquid has the best effect when being prepared, and can also be stored for at least two weeks under the refrigeration condition of 4 ℃.
7. The method of claim 4, wherein the blocking solution is applied to immunohistochemistry, and the method comprises the following steps: when the sealing liquid is used, the sealing time is 15min-24h, and the sealing temperature is 4-40 ℃.
8. The method of claim 4, wherein the blocking solution is applied to immunohistochemistry, and the method comprises the following steps: when the sealing liquid is used, the optimal sealing conditions are as follows: blocking at 37 ℃ for 30 min.
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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 |
| CN113552362A (en) * | 2021-07-23 | 2021-10-26 | 湖北百奥斯生物科技有限公司 | Novel immunofluorescence kit of signal amplification |
| 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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| 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 |
| CN113552362A (en) * | 2021-07-23 | 2021-10-26 | 湖北百奥斯生物科技有限公司 | Novel immunofluorescence kit of signal amplification |
| 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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