CN116381240A - Double-dyeing immunohistochemical kit and application - Google Patents
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Abstract
The invention provides a double-dyeing immunohistochemical kit and application thereof, and relates to the field of immunohistochemistry. Wherein, double-staining immunohistochemical kit includes: the kit comprises a fixing solution, a membrane rupture agent, a primary antibody, a secondary antibody and a staining agent, wherein the primary antibody comprises an LEF1 antibody and a CD20 antibody. The secondary antibody reagent comprises alkaline phosphatase AP labeled secondary antibody polymer and horseradish peroxidase HRP labeled secondary antibody polymer. The kit has complete cell morphology of the dyed sample, simple flow, and can effectively improve the identification degree of the cell morphology and the diagnosis accuracy.
Description
Technical Field
The invention relates to the field of immunohistochemistry, in particular to a double-dyeing immunohistochemical kit and application thereof.
Background
Chronic Lymphocytic Leukemia (CLL)/small lymphomas (SLL) are one of the common lymphoproliferative disorders in the adult patient population, manifested by monoclonal B-lymphocytosis in the peripheral blood or bone marrow. Diagnosis of CLL is largely dependent on immunophenotyping and immunohistochemistry, with most CLL patient cells expressing CD5 and B cell antigens CD19, CD20 and CD23. Typical CLL immunophenotypes are cd5+, cd23+, cd43+/-, CD10-, cd19+, CD20 dim (CD 20 weakly expressed), ig dim (ig weakly expressed). Some CLL patients may show a weak positive for ig bright (strong expression of ig), CD23-/dim, FMC 7.
Currently, B cell clonality is confirmed by staining mainly bone marrow biopsy specimen sections. However, it was found that Mantle Cell Lymphoma (MCL) immune expression type is cd5+, CD20 expression, and MCL patients sometimes have an immune phenotype similar to CLL, so that it is necessary to introduce a new diagnostic marker to realize differential diagnosis of CLL/SLL and MCL.
In addition, during the diagnosis of CLL/SLL, it is necessary to stain a bone marrow biopsy specimen slice, and since a large amount of bone mass exists in the bone marrow biopsy specimen, it is impossible to directly slice. Weak acid or strong acid is needed for decalcification treatment, and the solvents can cause certain damage to antigens, so false positives and false negatives are easy to occur, and inaccurate results are caused.
Disclosure of Invention
In order to solve the technical problems, the invention provides a double-dyeing immunohistochemical kit and application thereof.
The invention solves the technical problems by adopting the following technical scheme.
In a first aspect the invention provides a double-dye immunohistochemical kit comprising: the kit comprises a fixing solution, a membrane breaker, a primary antibody, a secondary antibody and a staining agent, wherein the primary antibody comprises an LEF1 antibody and a CD20 antibody; the secondary antibody reagent comprises alkaline phosphatase AP marked secondary antibody polymer and horseradish peroxidase HRP marked secondary antibody polymer.
In an exemplary embodiment of the invention, the LEF1 antibody and the CD20 antibody are derived from animals of two different species.
In an exemplary embodiment of the invention, the alkaline phosphatase AP labeled secondary antibody polymer is for binding to a primary antibody derived from a first species of animal, and the horseradish peroxidase HRP labeled secondary antibody polymer is for binding to a primary antibody derived from a second species of animal, the first species of animal being different from the second species of animal.
In an exemplary embodiment of the invention, the first and second animals are identical to animal species of the LEF1 antibody and the CD20 antibody, respectively.
In an exemplary embodiment of the present invention, the stain includes a peroxidase substrate stain, an alkaline phosphatase substrate stain, and a counterstain stain, the counterstain stain not masking the color development of the peroxidase substrate stain and alkaline phosphatase substrate stain.
In an exemplary embodiment of the present invention, the fixing liquid is a mixed liquid of methanol and formaldehyde.
In an exemplary embodiment of the invention, the double-dye immunohistochemical kit further comprises a protein blocker selected from one or more of bovine serum albumin buffer solution, goat serum buffer solution, horse serum buffer solution and casein buffer solution.
In an exemplary embodiment of the invention, the membrane breaker is selected from the group consisting of saponin, triton-X, tween-20, aldehydes, alcohols, ketones, sodium chloride, calcium chloride, sodium benzoate, disodium edetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium benzoate, polyoxyethylene nonylphenol ether, triton X-100, triton-X, and Tween-20 in PBS solution.
In an exemplary embodiment of the invention, the double-staining immunohistochemical kit is used for staining peripheral blood smears, bone marrow smears, cell smears.
In a second aspect, the invention provides a staining method based on LEF1/CD20 double staining, using a double staining immunohistochemical kit as described in any one of the above.
In an exemplary embodiment of the present invention, a dyeing method includes:
step S1, dripping a fixing liquid on a smear for fixing to obtain a sample;
step S2, dripping the protein blocking agent on the sample, and incubating to obtain a blocking sample;
step S3, dropwise adding a membrane breaker on the blocking sample, and incubating for a period of time;
step S4, adding the primary antibody reagent, and cleaning after incubation;
step S5, adding the secondary antibody reagent, and cleaning after incubation;
and S6, dropwise adding a peroxidase substrate staining solution, an alkaline phosphatase substrate staining solution and a hematoxylin staining solution for staining treatment to obtain a staining sample.
In an exemplary embodiment of the present invention, in step S6, the step of dyeing includes: firstly, dropwise adding horseradish enzyme substrate staining solution, incubating and cleaning, then dropwise adding alkaline phosphatase substrate staining solution, incubating and cleaning, and finally dropwise adding counterstain staining solution, incubating and cleaning to obtain a staining sample; or alternatively, the process may be performed,
firstly, dropwise adding alkaline phosphatase substrate staining solution, incubating and cleaning, then dropwise adding horseradish enzyme substrate staining solution, incubating and cleaning, and finally dropwise adding counterstain staining solution, incubating and cleaning to obtain a staining sample.
In a third aspect, the invention provides the use of a double-dye immunohistochemical kit according to any one of the preceding claims in the diagnosis of chronic lymphocytic leukaemia.
Compared with the prior art, the double-dyeing immunohistochemical kit, the dyeing method and the application have the beneficial effects that:
the double-dyeing immunohistochemical kit provided by the invention can be applied to auxiliary diagnosis of chronic lymphocytic leukemia, and can effectively improve the diagnosis accuracy. For CLL, both LFE1 (lymphokine 1) and CD20 are expressed on monoclonal B lymphocytes. For MCL, since LEF1 is expressed predominantly on CD3 positive T lymphocytes, there is little positive on CD20 positive monoclonal B lymphocytes. According to the invention, by performing double-staining treatment on LEF1/CD20, the expression of LEF1 is stained through cell nuclei, the expression of CD20 is stained through cell plasma, two different colors are used for positive double-staining, the positions of the two cells are respectively obtained, the abnormal expression of B lymphocytes can be more clearly defined, the interference of T cells is eliminated, and the differential diagnosis of CLL and MCL is realized. The double-dyeing immunohistochemical kit uses LEF1 antibody and CD20 antibody for double-dyeing, has strong specificity, and the dyeing method is simple and easy to realize, and can realize the whole dyeing process through automatic dyeing equipment. The invention has clear dyeing result and easy discrimination of cell morphology.
In addition, the staining method of the embodiment can be applied to peripheral blood smears, and the specimen application range is wide. Pretreatment procedures such as decalcification treatment and the like are not needed, so that antigen damage is effectively avoided, and the occurrence of false positive and false negative conditions is reduced. The sample is fixed by the fixing liquid, so that the cell morphology integrity is high.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a dyeing method provided by an embodiment of the invention;
FIG. 2 is a graph showing the result of staining of sample A provided in example 1 of the present invention;
FIG. 3 is a graph showing the result of staining of sample B provided in example 1 of the present invention;
fig. 4 is a graph of the staining results of sample C provided in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. It is to be understood that unless otherwise defined, all technical and scientific terms of this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in this patent is consistent with the description of the terminology in the documents given above, as defined generally in the art for the most part in the documents below.
The double-staining immunohistochemical kit, the staining method and the application of the embodiment of the present disclosure are specifically described below.
The double-staining immunohistochemical kit provided by the embodiment of the disclosure comprises: fixing solution, a membrane breaker, a primary antibody reagent, a secondary antibody reagent and a staining agent.
In an embodiment of the invention, the double-dyeing immunohistochemical kit further comprises a protein blocking agent. For example, bovine Serum Albumin (BSA) and the like can be used as the protein blocking agent. During immunohistochemical staining, the sample may be subjected to some interfering substances, such as endogenous antibodies, other binding proteins, autoantibodies, etc. Interferents are capable of interfering with the reaction between an antibody and an analyte through non-specific binding, resulting in false positives or false negatives. The protein blocking agent can effectively block the interference in the test and provide the test accuracy. In one embodiment, the protein blocker may also include a stabilizer, surfactant, or the like. The stabilizer may be, for example, sodium chloride, a surfactant such as tween 20, or the like.
Further preferably, in other embodiments, the protein blocker may be selected from one or more of a bovine serum protein buffer, a goat serum buffer, a horse serum buffer, and a casein buffer. In particular, the protein blocker may be, for example, a bovine serum albumin buffer solution or a mixture of a bovine serum albumin buffer solution and a casein buffer solution. The protein blocker may also be a commercially available product, and the present disclosure is not particularly limited.
In a preferred embodiment of the present disclosure, the fixing solution is a mixed solution of methanol and formaldehyde, specifically, the fixing solution is a mixed solution of methanol and formaldehyde in a mass ratio of 1:0.5-2. More preferably, the fixing liquid is a mixed liquid of methanol and formaldehyde with a mass ratio of 1:0.9-1.1. For example, in a preferred embodiment of the present disclosure, the fixing liquid is methanol and formaldehyde in a mass ratio of 1:1. The fixing liquid can effectively fix cells on the premise of ensuring the antigen activity, improves the integrity of cell morphology, and is favorable for the subsequent dyeing process and cell morphology recognition after dyeing.
In a preferred embodiment of the present invention, the membrane breaker is selected from the group consisting of saponins, triton-X and Tween-20, aldehydes, alcohols, ketones, sodium chloride, calcium chloride, sodium benzoate, disodium edetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium benzoate, polyoxyethylene nonylphenol ether, triton-X-100, triton-X and Tween-20 in PBS. By applying the membrane breaker, a perforating operation is performed on the cell matrix, so that subsequent antibodies can enter the cell to bind with the antigen.
Further preferably, the breaker is preferably a BD breaker. For example, commercially available BD Phosflow is selected TM Perm Buffer II rupture of membranes (cat# 558052) or BD Photoflow TM Perm Buffer II rupture of membranes (cat# 558050), etc.
In an embodiment of the present invention, the primary antibody includes an LEF1 antibody, a CD20 antibody. Specifically, the LEF1 antibody and CD20 antibody are derived from animals of two different species. For example, in one embodiment, the LEF1 antibody is a rabbit anti-human LEF1 monoclonal antibody (clone number BP6113, toyo Biomedicine Co., ltd.). In one embodiment, the CD20 antibody is a murine monoclonal antibody (CD 20 mouse Monoclonal Antibody, clone number L26) (bi yun tian biotechnology).
In other embodiments, LFE1 may be a murine monoclonal antibody and CD20 may be a rabbit monoclonal antibody. Other commercial products can be selected for the LEF1 antibody and the CD20 antibody in this embodiment, as long as one of the LEF1 antibody and the CD20 antibody is ensured to be rabbit antibody, and the other is murine antibody, and the invention is not particularly limited.
In an embodiment of the invention, the secondary antibody reagent comprises an alkaline phosphatase AP-labeled secondary antibody polymer and a horseradish peroxidase HRP-labeled secondary antibody polymer. Specifically, the alkaline phosphatase AP labeled secondary antibody polymer is used for binding to a primary antibody derived from a first species of animal, and the horseradish peroxidase HRP labeled secondary antibody polymer is used for binding to a primary antibody derived from a second species of animal, the first species of animal being different from the second species of animal.
For example, in one embodiment, commercial products such as alkaline phosphatase AP-labeled affinity purified goat anti-rabbit IgG (H+L) secondary antibodies, HRP-labeled goat anti-mouse IgG F (ab') 2 secondary antibodies, and the like, manufactured by Eimer technology, may be selected.
It should be noted that, the alkaline phosphatase AP-labeled secondary antibody polymer and the horseradish peroxidase HRP-labeled secondary antibody polymer may be other commercial products, for example, one of them is anti-rabbit polymer and the other is anti-mouse polymer.
In an embodiment of the invention, the alkaline phosphatase AP-labeled secondary antibody polymer and the horseradish peroxidase HRP-labeled secondary antibody polymer are identical to animal species of the LEF1 antibody and the CD20 antibody, respectively. For example, LEF1 is a murine antibody, CD20 antibody is a rabbit antibody, alkaline phosphatase AP-labeled secondary antibody is an anti-murine polymer, and horseradish peroxidase HRP-labeled secondary antibody is an anti-rabbit polymer.
In the immunohistochemical detection process, the primary antibody specifically binds to a target protein (antigen), and the primary antibody is identified through the secondary antibody, so that the signal amplification effect is provided for detection.
In a preferred embodiment of the invention, the stain comprises a peroxidase substrate stain, an alkaline phosphatase substrate stain, and a counterstain stain. Preferably, in the examples to be given, the peroxygenase substrate staining solution may be DAB staining solution, and the alkaline phosphatase substrate staining solution may be fast red staining solution. The development of the counterstain dye solution does not mask the development of the peroxidase substrate dye solution and alkaline phosphatase substrate dye solution
In one embodiment, the DAB dye solution is commercially available and comprises DAB (3, 3-diaminobenzidine hydrochloride) solution and H 2 O 2 . The horseradish peroxidase HRP-marked secondary antibody polymer can catalyze and decompose hydrogen peroxide in DAB staining solution into high-activity hydroxyl radical molecules, oxidize diaminobenzidine and form brown yellow precipitate, so that positioning is realized.
In one embodiment, the fast red staining solution may be obtained from commercial products, the components of which include alkaline phosphatase substrate solution and fast red solution. Specifically, the alkaline phosphatase substrate solution is fast red (fast red), and the fast red solution comprises naphthol phosphate and tris. In the dyeing process, the Alkaline Phosphatase (AP) marked secondary antibody polymer can catalyze the hydrolysis of phosphate groups of alkaline phosphatase substrates to form phosphate non-naphthol, and the phosphate non-naphthol reacts with solid red solution to generate insoluble red product precipitate at the position of the alkaline phosphatase, so that positioning is realized.
In one embodiment, the counterstain is a hematoxylin stain or a methyl green stain. Hematoxylin staining solutions and methyl green staining solutions can be obtained by commercial products. Counterstaining is performed by hematoxylin staining solution, and the cell nuclei are stained blue-violet with hematoxylin, or green with methyl green staining solution. The cytoplasma, the fiber and the like are not dyed, the effect of setting off the whole cell distribution and the tissue morphology is achieved, and the observation and the recognition are easier.
In the embodiment of the invention, the double-staining immunohistochemical kit can be used for staining peripheral blood smears, bone marrow smears and cell smears, and has clear staining results and wide application range. More preferably, the double-staining immunohistochemical kit is applied to the bone marrow peripheral blood smear, and the bone marrow peripheral blood smear does not need to carry out a complex pretreatment process, thereby greatly saving pretreatment time and avoiding damage to cells caused by decalcification and other treatments.
As shown in FIG. 1, the embodiment of the invention also provides a staining method based on LEF1/CD20 double staining, and the double staining immunohistochemical kit is adopted. Specifically, in an embodiment of the present invention, a dyeing method includes:
step S1, dripping a fixing liquid on a smear for fixing to obtain a sample;
step S2, dripping the protein blocking agent on the sample, and incubating to obtain a blocking sample;
step S3, dropwise adding a membrane breaker on the blocking sample, and incubating for a period of time;
step S4, adding the primary antibody reagent, and cleaning after incubation;
step S5, adding the secondary antibody reagent, and cleaning after incubation;
and S6, dropwise adding a peroxidase substrate staining solution, an alkaline phosphatase substrate staining solution and a hematoxylin staining solution for staining treatment to obtain a staining sample.
Specifically, in step S1, the fixing solution is preferably methanol and formaldehyde in a mass ratio of 1:1. The fixing time is 1-30 min. Preferably, in one embodiment, the fixed time is 1min. The fixing step can effectively improve the integrity of cell morphology on the premise of ensuring the activity of the antigen.
Step S2, dripping the protein blocking agent on the sample, and incubating to obtain a blocking sample.
Specifically, in this step, the protein block is one or more of primary anti-dilution, secondary anti-dilution, bovine serum protein solution, goat serum solution, horse serum solution, and casein solution. Particularly, when the kit is applied to a bone marrow peripheral blood smear, the sample contains more dry stains, and the detection accuracy can be effectively improved by adding a protein blocking agent.
Further, in this step, the protein blocking agent may be added dropwise in the range of 50 to 200. Mu.l, and the incubation time may be in the range of 1 to 60 minutes. Further, the incubation time is preferably 10min, and the incubation temperature is 15-35 ℃. And spin-drying is performed after incubation is completed, and cleaning is not needed.
And step S3, dropwise adding 50-200 microliters of the membrane breaker solution to the blocking sample, wherein the concentration of the membrane breaker solution is 5% -35%, and incubating for a period of time. Specifically, in one embodiment, the membrane breaker solution contains sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, polyoxyethylene nonylphenol ether, triton X-100, and paraformaldehyde.
Further preferably, the breaker is a flow breaker manufactured by BD company. After the membrane breaker is added, the incubation time is 1-60 min, and further preferably, the incubation time is 10min, and the incubation temperature is 15-35 ℃. Through this step, holes can be punched in the cells, facilitating the binding of the primary antibody.
And S4, adding the primary antibody reagent into the sample obtained in the step S3, and washing with PBS buffer solution after incubation. Specifically, in this step, the primary antibody is added dropwise, and the specific amount of the primary antibody may be selected according to the specifications of the specifically purchased primary antibody, and the present disclosure is not particularly limited. After adding the primary antibody, the smear can be washed by PBS buffer solution after incubating for 3-800 min at 4-60 ℃. The PBS buffer solution may be washed 3min each time, and repeatedly washed 3 times. It will be appreciated that the incubation time after the primary antibody is added may be adjusted to different incubation temperatures, for example, in a preferred embodiment, between 20 and 40 minutes at 30 to 40 ℃. Further, in this step, the incubation temperature was 35℃and the incubation time was 30min. The incubation time is shorter under the condition, and the incubation effect is better.
It is noted that the present disclosure uses CD20 antibody and LEF1 antibody as primary antibody, and in one embodiment, the CD20 antibody and LEF1 antibody may be added simultaneously for incubation. In other embodiments, the CD20 antibody may be added for incubation, followed by the LEF1 antibody; or adding LEF1 antibody for incubation, and then adding CD20 antibody for incubation.
And S5, adding a secondary antibody reagent, incubating for 5-800 min at the temperature of 4-60 ℃, and cleaning for multiple times by adopting PBS buffer solution. . The amount of the secondary antibody may be determined according to a specific manufacturer, and may be, for example, 135u of the secondary antibody purchased from Xiaomentung biomedical technologies Co. It will be appreciated that the incubation time after the primary antibody reagent is added may be adjusted to different incubation temperatures, for example, in a preferred embodiment, between 10 and 40 minutes at 20 and 45℃after the secondary antibody reagent is added. The incubation time is shorter under the condition, and the incubation effect is better.
In this step, in one embodiment, the alkaline phosphatase AP-labeled secondary antibody polymer and the horseradish peroxidase HRP-labeled secondary antibody polymer may be simultaneously added for incubation. In other embodiments, the secondary antibody polymer marked by alkaline phosphatase AP and the secondary antibody polymer marked by horseradish peroxidase HRP can be added separately for incubation, the secondary antibody polymer marked by alkaline phosphatase AP can be added first for incubation, and the secondary antibody polymer marked by horseradish peroxidase HRP can be added first for incubation.
And S6, dropwise adding a peroxidase substrate staining solution, an alkaline phosphatase substrate staining solution and a hematoxylin staining solution for staining treatment to obtain a staining sample.
In one embodiment, step S6 specifically includes: firstly, dropwise adding a horseradish enzyme substrate staining solution, incubating and cleaning, then dropwise adding an alkaline phosphatase substrate staining solution, incubating and cleaning, and finally dropwise adding a hematoxylin staining solution, incubating and cleaning to obtain a staining sample. Specifically, in one embodiment, DAB staining solution is firstly dripped, incubated at 5-50 ℃ for 3-120 min, and then washed with pure water for 2 times, each time for 2min. Then dripping the accelerated red staining solution, incubating for 3-120 min at 5-50 ℃, and then washing with pure water for 2 times, each time for 2min. Then 135ul hematoxylin staining solution is dripped into the smear, and after incubation is carried out for 3-120 min at 5-50 ℃, the smear is washed with pure water for 2 times, and each washing is carried out for 2min.
In another embodiment, step S6 specifically includes: firstly, dropwise adding alkaline phosphatase substrate staining solution, incubating and cleaning, then dropwise adding horseradish enzyme substrate staining solution, incubating and cleaning, and finally dropwise adding hematoxylin staining solution, incubating and cleaning to obtain a staining sample. Specifically, in one embodiment, the accelerated red staining solution is dripped first, incubated at 5-50 ℃ for 3-120 min, and then washed with pure water for 2 times, each for 2min. Then dripping DAB staining solution, incubating for 3-120 min at 5-50 ℃, and washing with pure water for 2 times, each time for 2min. Then 135ul hematoxylin staining solution is dripped into the smear, and after incubation is carried out for 3-120 min at 5-50 ℃, the smear is washed with pure water for 2 times, and each washing is carried out for 2min.
The embodiment of the invention also provides application of the double-dyeing immunohistochemical kit in diagnosis of chronic lymphocytic leukemia.
The features and capabilities of the present disclosure are described in further detail below in connection with the examples.
Example 1
Reagent:
the following samples A, B, C were subjected to a staining treatment:
sample a: peripheral blood smears of CLL patients diagnosed in a hospital;
sample B: negative control: normal T lymphocyte smear;
sample C: peripheral blood smears of MCL patients were diagnosed in a hospital.
The dyeing process is as follows:
(1) Fixing: dripping the fixed liquid onto a sample for fixing for 1min;
(2) Blocking: dripping 40 μl of protein blocker, incubating at room temperature for 10min, and spin-drying (without cleaning);
(3) Rupture of membranes: dripping 150 μl of membrane breaker solvent, incubating at room temperature for 10min, and covering blood membrane;
(4) Incubation resistance: after dropping 135. Mu.l of primary antibody reagent and incubating at 35℃for 30min, the sections were washed 3 times with PBS buffer for 3min each.
(5) Secondary antibody incubation: after dropping 135. Mu.l of the secondary antibody reagent and incubating for 18min at 35 ℃, the sections were washed with PBS buffer for 3 times each for 3min.
(6) Color development: 180 μl DAB staining solution was added dropwise to the smear, and after incubation at 35℃for 6min, the smear was washed 2 times with pure water for 2min each. The red staining solution was added dropwise in an amount of 150. Mu.l, and incubated for 20min.
(7) Counterstaining: the smear was added dropwise with 135. Mu.l of hematoxylin staining solution, incubated at 35℃for 4min, and then washed with pure water 2 times for 2min each.
(8) And (5) microscopic examination: after acidification, bluing, examination under a microscope.
Fig. 2 shows the result of the staining of sample a, fig. 3 shows the result of the staining of sample B, and fig. 4 shows the result of the staining of sample C.
As can be seen from the staining results, in fig. 2, the brown color of the envelope indicated CD20 positive, the red color of the nucleus indicated LEF1 positive, CD20 co-positive with LEF1, and identified as CLL cells. In fig. 3, no detected membrane brown indicated CD20 negative, nuclear red indicated LEF1 positive, identified as normal T lymphocytes. In fig. 4, the brown color of the envelope indicated CD20 positive, the undetectable nuclear red color indicated LEF1 negative, and MCL cells were identified. As can be seen, the staining method of the present disclosure is able to effectively distinguish CLL from MCL. The staining chart shows that the stained cells have clear morphology, clear cell nucleus, cell membrane and cytoplasm, accurate positive location and clear signal expression, and can be applied to the auxiliary diagnosis of the sexual lymphocytic leukemia.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. 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.
Claims (10)
1. A double-dye immunohistochemical kit comprising: the kit comprises a fixing solution, a membrane breaker, a primary antibody, a secondary antibody and a staining agent, wherein the primary antibody comprises an LEF1 antibody and a CD20 antibody; the secondary antibody reagent comprises alkaline phosphatase AP marked secondary antibody polymer and horseradish peroxidase HRP marked secondary antibody polymer.
2. The double-dye immunohistochemical kit according to claim 1, wherein the LEF1 antibody and the CD20 antibody are derived from animals of two different species.
3. The double-dye immunohistochemical kit according to claim 1, wherein the alkaline phosphatase AP-labeled secondary antibody polymer is used for binding to a primary antibody derived from a first species of animal, and the horseradish peroxidase HRP-labeled secondary antibody polymer is used for binding to a primary antibody derived from a second species of animal, the first species of animal and the second species of animal being different.
4. The double-dyeing immunohistochemical kit according to claim 1, wherein the first species of animal and the second species of animal are identical to the animal species of the LEF1 antibody and the CD20 antibody, respectively.
5. The double-dyeing immunohistochemical kit according to claim 1, wherein the staining agent comprises a peroxidase substrate staining solution, an alkaline phosphatase substrate staining solution and a counterstain staining solution, and the color development of the counterstain staining solution does not mask the color development of the peroxidase substrate staining solution and the alkaline phosphatase substrate staining solution.
6. The double-dye immunohistochemical kit according to claim 1, wherein the fixing solution is a mixed solution of methanol and formaldehyde.
7. The double-dye immunohistochemical kit according to claim 1 further comprising a protein blocker selected from one or more of bovine serum albumin buffer, goat serum buffer, horse serum buffer and casein phosphate buffer.
8. The double-dye immunohistochemical kit according to claim 1, wherein the membrane breaker is selected from one or more of saponins, aldehydes, alcohols, ketones, sodium chloride, calcium chloride, sodium benzoate, disodium edetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium benzoate, polyoxyethylene nonylphenol ether, triton X-100, triton-X and Tween-20.
9. The double-staining immunohistochemical kit of claim 1, wherein the double-staining immunohistochemical kit is used for staining peripheral blood smears, bone marrow smears, cell smears.
10. Use of a double-dye immunohistochemical kit according to any one of claims 1 to 9 in the diagnosis of chronic lymphocytic leukemia.
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| CN117233393B (en) * | 2023-11-15 | 2024-02-09 | 四川大学华西医院 | Double-immunohistochemical staining kit and application thereof in identifying benign and malignant bile duct epithelial tumors |
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