Method for simultaneously carrying out EBER and CD56 mark staining on sample slices

Abstract

The invention discloses a method for simultaneously carrying out EBER and CD56 mark staining on a sample section. The method comprises the following steps: sequentially carrying out section baking treatment, dewaxing hydration treatment, endogenous enzyme closing operation and antigen repair on the sample section; carrying out in-situ hybridization on the treated sample slices, sequentially carrying out primary antibody incubation and AP secondary antibody incubation, then carrying out red dyeing substrate color development, and observing a color development result; and carrying out HRP secondary antibody incubation on the sample section after the red staining substrate is subjected to color development, cleaning with a cleaning solution, carrying out CD56 labeling staining, and observing the staining result of the section. By synchronous operation of EBER in-situ hybridization and CD56 immunohistochemical staining, observation of two staining results on the same sample is realized, experiment efficiency and result reliability are improved, false negative results of EBER detection caused by a traditional method are avoided, and detection accuracy is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pathological diagnosis, and particularly relates to a method for simultaneously performing EBER and CD56 labeling and staining on a sample section. Background Art

[0002] Immunohistochemical staining refers to binding a fluorescent or color-developable chemical substance to an antibody, and using the specific binding reaction between an antigen and an antibody in immunological principles to detect the presence of a target antigen in cells or tissues. A pathologist can observe the stained pathological section under a microscope to observe abnormal cell expression, so as to determine whether a patient has a certain malignant tumor and the type and differentiation of the tumor.

[0003] EBER in-situ hybridization is a method for detecting the expression and localization of a small mRNA molecule encoded by Epstein-Barr virus (EBV) in cells. Using a probe complementary to the RNA sequence to generate a hybridization reaction with the target RNA molecule to detect the presence and spatial position of the RNA molecule in cells. EBER in-situ hybridization has accurate localization and relatively high sensitivity, and is closely related to tissue morphology, so it is an important method for pathological diagnosis.

[0004] Aggressive NK cell leukemia (ANKL) is a fulminant disease with extremely poor prognosis. Patients usually present with acute fever, systemic symptoms, hepatosplenomegaly and disseminated intravascular coagulation, often accompanied by hemophagocytic lymphohistiocytosis (HLH). Morphological and immunological features are crucial for the diagnosis of ANKL. Bone marrow biopsy can comprehensively evaluate the morphological manifestations of tissues and immunohistochemical markers of cells to better identify aggressive tumor cells. The strong correlation with EBV indicates the pathogenic role of this virus. CD56 immunohistochemistry can improve the detection sensitivity, and CD56 is positive in most ANKL cases.

[0005] Currently, EBER in-situ hybridization is mostly performed in lymph nodes and is difficult to detect in bone marrow biopsy samples because bone marrow biopsy samples often need to be treated with strong acid for decalcification, which will cause a certain degree of damage to nucleic acids. Traditional methods will result in false negatives in EBER detection. We have tried the reagents of many immunohistochemistry companies on the market, and all EBER detections are false negatives. At the same time, according to the existing technology, only EBER and CD56 labeling and staining can be performed on samples separately, and it is impossible to conveniently / accurately / quickly observe the co-distribution of the two labels in tumor cells. Therefore, an improved staining method is needed. Summary of the Invention

[0006] The object of the present invention is to address the deficiencies of the prior art. The present invention provides an improved staining method, which solves the problems that EBER in situ hybridization is mostly carried out in lymph nodes and is difficult to detect in bone marrow biopsy samples. Because bone marrow biopsy samples often need to be treated with strong acid decalcification, which will cause a certain degree of damage to nucleic acids, and only EBER and CD56 staining can be performed on the samples separately, and it is impossible to conveniently / accurately / quickly observe the co-distribution of the two stains in tumor cells.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A method for simultaneously performing EBER and CD56 labeled staining on a sample section, comprising the following steps:

[0008] (1) Sequentially perform baking treatment, dewaxing and hydration treatment, blocking of endogenous enzymes operation and antigen repair on the sample section;

[0009] (2) Perform in situ hybridization on the treated sample section, sequentially perform primary antibody incubation and AP secondary antibody incubation, and then perform red staining substrate color development, and observe the color development result;

[0010] (3) Perform HRP secondary antibody incubation on the sample section after red staining substrate color development, wash with a washing solution, then perform CD56 labeled staining, and then perform coverslipping treatment, and observe the staining result of the section.

[0011] Further, the temperature of the baking treatment in step (1) is 60-65 °C, and the time is 60-90 min; the dewaxing and hydration treatment is to sequentially soak the sample section in xylene solution for 5-10 min × 3 times, and then soak in gradient alcohol for 2-3 min each time. The soaked section is rinsed with pure water to remove the residual alcohol on the surface, and rinsed repeatedly for 30-60 s × 3 times to complete dewaxing and hydration of the sample; the gradient alcohol concentrations are 100%, 100%, 95%, and 75% in descending order.

[0012] Further, the operation of blocking endogenous enzymes in step (1) is to soak the tissue section in a blocking agent for 5-15 min; the antigen repair is to heat the tissue section in an immunohistochemical antigen repair buffer, cool it, soak it in pure water, shake off the liquid on the slide, draw a water-blocking area along the edge of the tissue section with an immunohistochemical pen, and then soak the sample in a washing solution for 2-3 min × 3 times.

[0013] Further, the in situ hybridization in step (2) is to drop an EBER probe on the tissue section, cover with a coverslip, and then use an in situ hybridization mounting agent to mount the sample, place it in an in situ hybridization instrument for hybridization, the hybridization temperature is 40-45 °C, and the time is 90-100 min. After completion, soak it in a washing solution for 2-3 min × 3 times.

[0014] Furthermore, in step (2), for the primary antibody incubation and AP secondary antibody incubation, digoxin and CD56 antibody reagents are dropped onto the sample sections and incubated in a wet box to complete the primary antibody incubation. Subsequently, the samples are soaked in the cleaning solution for 2 - 3 min × 3 times. After soaking and discarding the cleaning solution, AP secondary antibody is dropped respectively and incubated at room temperature to complete the AP secondary antibody incubation.

[0015] Furthermore, in the red staining substrate color development in step (2), the sample sections are soaked in the cleaning solution for 2 - 3 min × 3 times. After soaking and discarding the cleaning solution, AP-Red chromogenic solution is dropped for staining, and the staining time is 20 - 25 min.

[0016] Furthermore, in the HRP secondary antibody incubation in step (3), the sample sections are soaked in the cleaning solution for 2 - 3 min × 3 times. After soaking and discarding the cleaning solution, HRP secondary antibody is dropped and incubated at room temperature for 15 - 20 min.

[0017] Furthermore, in the CD56 labeling staining and mounting in step (3), DAB staining solution is dropped onto the sample sections to completely cover the specimens and stained for 3 - 5 min. The samples are rinsed with pure water for 30 - 60 s × 1 time, and the excess water is discarded. Then hematoxylin staining solution is dropped to completely cover the specimens for staining. Subsequently, the samples are rinsed with pure water for 30 - 60 s to wash away the excess staining solution, and then soaked in the immunohistochemical antigen repair buffer for 2 - 3 min to bluing; the sample sections are soaked in gradient alcohol for 2 - 3 min each in turn, and mounted with a fragrant mounting agent. The mounted sections are placed on a drying plate to complete the dehydration, clearing and mounting process.

[0018] Furthermore, the gradient alcohol concentrations are 100%, 100%, 95%, 75% from high to low in turn.

[0019] Furthermore, the staining time for dropping hematoxylin staining solution for staining is 1 - 2 min.

[0020] The present invention provides an improved staining method. It has the following beneficial effects:

[0021] 1. The present invention synchronizes the EBER in situ hybridization and CD56 immunohistochemical staining, realizes the observation of two staining results on the same sample, improves the experimental efficiency and the reliability of the results, avoids the false negative results of EBER detection caused by the traditional method, and improves the detection accuracy.

[0022] 2. The present invention simplifies the operation process, makes the experimental steps more standardized and easy to operate, and is applicable to various types of samples, including difficult-to-process bone marrow biopsy samples. This method not only saves time and resources, but also expands the application range of the staining technology and improves the adaptability to different sample types. Brief Description of the Drawings

[0023] Figure 1 It is a flowchart of the staining method in the embodiment.

[0024] Figure 2 It is a double-label staining diagram of EBER (a) and CD56 (b) of the bone marrow trephine biopsy specimen in the embodiment. Detailed Embodiments

[0025] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0026] Embodiment:

[0027] Please refer to the attached Figure 1 , the embodiment of the present invention provides an improved staining method, including the following steps:

[0028] Step 1: Bake the sample section at 60-65 °C for 60-90 min continuously. Immerse the tissue section in xylene solution for 5-10 min × 3 times respectively, and then immerse it in gradient alcohol for 2-3 min each time (the gradient alcohol concentrations are 100% - 100% - 95% - 75% from high to low). After immersion, rinse the surface of the section with pure water to remove the residual alcohol, and repeat the rinsing 30-60 s × 3 times to complete dewaxing and hydration of the sample.

[0029] Step 2: Immerse the tissue section in the blocking agent for 5-15 min and rinse it with pure water for 30-60 s to complete the operation of blocking endogenous enzymes. Then start the antigen repair process. Pour the immunohistochemical antigen repair buffer into the box, boil it, continue heating for 20-30 min, stop heating after completion, and let it cool naturally. Then soak the sample in pure water for 2-3 min. After soaking, shake off the liquid on the slide, and then blot the liquid at the edge of the tissue block with absorbent paper. Use an immunohistochemical pen to draw a water-blocking area along the edge of the tissue section. Then soak the sample in the cleaning solution for 2-3 min × 3 times.

[0030] Step 3: Perform in situ hybridization. Drop about 20 μL of EBER probe, cover with a cover slip, and then use an in situ hybridization sealing agent to seal the sample. Place it in an in situ hybridization instrument for hybridization, select an environment at 40-45 °C for 90-100 min. After completion, soak it in the cleaning solution for 2-3 min × 3 times.

[0031] Step 4: Add appropriate amounts of digoxin and CD56 antibody reagents to the samples, incubate in a wet box for 20 - 30 min to complete the primary antibody incubation. Subsequently, soak the samples in the washing solution for 2 - 3 min × 3 times. After draining the washing solution after soaking, add appropriate amounts of AP secondary antibody respectively. The addition amount depends on the size of the section and should be sufficient to completely cover the specimen. Incubate at room temperature for 20 - 25 minutes to complete the AP secondary antibody incubation;

[0032] Step 5: Perform red staining substrate color development. Soak the sections in the washing solution for 2 - 3 min × 3 times. After draining the washing solution after soaking, add appropriate amounts of AP - Red chromogenic solution for staining, and the staining time is 20 - 25 min;

[0033] Step 6: Perform HRP secondary antibody incubation. Soak the sections in the washing solution for 2 - 3 min × 3 times. After draining the washing solution after soaking, add appropriate amounts of HRP secondary antibody respectively. The addition amount depends on the size of the section and should be sufficient to completely cover the specimen. Incubate at room temperature for 15 - 20 min;

[0034] Step 7: Soak the samples in the washing solution for 2 - 3 min × 3 times. After draining the washing solution after soaking, add appropriate amounts of DAB staining solution to completely cover the specimen and stain for 3 - 5 min. The staining time is adjusted temporarily according to the color development observed under the microscope;

[0035] Step 8: Perform counterstaining. Rinse the samples with pure water for 30 - 60 s × 1 time, drain the excess water, and add appropriate amounts of hematoxylin staining solution to completely cover the specimen for staining, lasting for 1 - 2 min. Subsequently, rinse the samples with pure water for 30 - 60 s to wash off the excess staining solution, and then soak the samples in the immunohistochemical antigen repair buffer for 2 - 3 min to blue back;

[0036] Step 9: Soak the sample sections in gradient alcohols for 2 - 3 min each in turn (the gradient alcohol concentrations are 100% - 100% - 95% - 75% from high to low). Use a fragrant mounting medium for mounting. Place the mounted sections on a drying plate to complete the dehydration, clearing, and mounting processes;

[0037] Step 10: Observe the staining results of the sections under the microscope.

[0038] Figure 2It is a double-label staining diagram of EBER (a) (×10) and CD56 (b) (×40) in a bone marrow trephine biopsy specimen. The nuclei of tumor cells in aggressive NK cell leukemia show red positive signals, indicating EBV infection, and the cytoplasm of tumor cells shows brown positive signals, confirming their NK cell phenotype. Through co-localization technology (EBER+ / CD56+), it is clear that the tumor cells are infected with EBV, supporting the direct role of EBV in tumorigenesis. At the same time, the high expression of EBER in tumor cells suggests that EBV may activate carcinogenic pathways such as NF-κB through latent infection (such as latent membrane proteins LMP1 / 2 or EBNA2), promoting the malignant proliferation of NK cells. Aggressive NK cell leukemia has a high incidence in the Asian population, which is consistent with the geographical prevalence of EBV, further supporting its etiological association. The EBER / CD56 double-label staining provides key etiological and immunophenotypic evidence for aggressive NK cell leukemia, strengthening the core position of EBV in the pathogenesis of this disease.

[0039] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for simultaneously staining a sample slice with EBER and CD56 markers, characterized in that The following steps are involved: (1) The sample sections are sequentially subjected to baking, dewaxing and hydration, endogenous enzyme blocking and antigen repair; (2) Perform in situ hybridization on the treated sample sections, incubate with primary antibody and AP secondary antibody in sequence, then develop with red-stained substrate and observe the color development results; (3) After the red-stained substrate is used for color development, the sample sections are incubated with HRP secondary antibodies, washed with cleaning solution, and then stained with CD56 markers. After that, the sections are sealed and the staining results are observed.

2. The method for simultaneously staining sample slices with EBER and CD56 markers according to claim 1, characterized in that: The temperature of the baking treatment in step (1) is 60-65°C and the time is 60-90 minutes. The dewaxing and hydrating treatment is to place the sample slices in a xylene solution and soak them for 5-10 minutes × 3 times, and then soak them in gradient alcohol for 2-3 minutes each. The slices that have been soaked are rinsed with pure water to remove the alcohol remaining on the surface, and the washing is repeated for 30-60 seconds × 3 times to complete the dewaxing and hydration of the samples. The gradient alcohol concentration is 100%, 100%, 95%, and 75% from high to low.

3. The method for simultaneously staining sample slices with EBER and CD56 markers according to claim 1, characterized in that: The blocking of endogenous enzymes in step (1) is to soak the tissue sections in a blocking agent for 5 to 15 minutes; the antigen repair is to heat the tissue sections in an immunohistochemical antigen repair buffer, soak them in pure water after cooling, shake off the liquid on the glass slide, use an immunohistochemical pen to mark the water-blocking area along the edge of the tissue sections, and then soak the samples in a cleaning solution for 2 to 3 minutes × 3 times.

4. The method for simultaneously staining sample slices with EBER and CD56 markers according to claim 1, characterized in that: The in situ hybridization in step (2) is to drop the EBER probe on the tissue section, add a coverslip and then use an in situ hybridization mounting medium to seal the sample, place it in an in situ hybridization instrument for hybridization, the hybridization temperature is 40-45°C, the time is 90-100 minutes, and after completion, soak it in a cleaning solution for 2-3 minutes × 3 times.

5. The method for simultaneously staining sample slices with EBER and CD56 markers according to claim 1, characterized in that: The primary antibody incubation and AP secondary antibody incubation in step (2) are to drip digoxin and CD56 antibody reagents onto the sample slices, incubate in a wet box, complete the primary antibody incubation, then soak the sample in a cleaning solution for 2 to 3 minutes × 3 times, after the soaking is completed and the cleaning solution is shaken off, AP secondary antibody is dripped on each slice, and incubated at room temperature to complete the AP secondary antibody incubation.

6. The method for simultaneously staining sample slices with EBER and CD56 markers according to claim 1, characterized in that: The red dye substrate color development in step (2) is to soak the sample slice in a cleaning solution for 2 to 3 minutes × 3 times, and after the soaking is completed and the cleaning solution is shaken off, AP-Red color development solution is added dropwise for staining, and the staining time is 20 to 25 minutes.

7. The method for simultaneously staining sample slices with EBER and CD56 markers according to claim 1, characterized in that: The HRP secondary antibody incubation in step (3) is to soak the sample slices in a cleaning solution for 2 to 3 minutes × 3 times, and after the soaking is completed and the cleaning solution is shaken off, the HRP secondary antibody is added dropwise and incubated at room temperature for 15 to 20 minutes.

8. The method for simultaneously staining sample slices with EBER and CD56 markers according to claim 1, characterized in that: The CD56 marker staining and sealing treatment in step (3) is to drip DAB staining solution on the sample slice to completely cover the specimen, and stain for 3 to 5 minutes. Rinse the sample with pure water for 30 to 60 seconds × 1 time, shake off excess water, and drip hematoxylin staining solution to completely cover the specimen for staining, then rinse the sample with pure water for 30 to 60 seconds to wash away excess staining solution, and then soak the sample in immunohistochemical antigen repair buffer for 2 to 3 minutes to return to blue; soak the sample slices in gradient alcohol for 2 to 3 minutes each, use scented sealing agent for sealing, and place the sealed slices on a drying plate to complete the dehydration, transparency and sealing process.

9. The method for simultaneously staining sample slices with EBER and CD56 markers according to claim 8, characterized in that: The gradient alcohol concentration is 100%, 100%, 95% and 75% from high to low.

10. The method for simultaneously staining sample slices with EBER and CD56 markers according to claim 8, characterized in that: The time for adding the hematoxylin staining solution for staining is 1 to 2 minutes.

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