A method for preparing cell pellets using mucin emulsifiers and applications thereof

Abstract

The application discloses a method for preparing a cell block by using mucin emulsifier and application thereof, and the preparation method comprises the following steps: centrifuging a non-gynecological sample to obtain a first separation liquid, adding the first separation liquid into a formaldehyde-alcohol solution to centrifuge to obtain a second separation liquid, and adding the second separation liquid into mucin emulsifier to centrifuge to obtain a cell block. The preparation method is simple, the manufacturing cost is low, the cell block has high enrichment efficiency, cell loss is small, and the mucin emulsifier does not affect cell activity. The cell block prepared by using the mucin emulsifier has the following advantages: the appearance of the section of the cell block is complete, the section has a regular circular structure, the porosity is moderate, and the thickness is uniform; the application effect of the section in two kinds of staining is good: the hematoxylin-eosin staining has a clear background and clear contrast, cells are uniformly distributed in a circular area, cell morphology presents a stereoscopic effect, and the excellent rate of the stained section is 98.94%; the immunohistochemical staining has a clean background, and structures and cells are specifically dyed, and the excellent rate of the stained section is 99.30%, so that the cell block has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a method for preparing cell blocks and its application, particularly a method for preparing cell blocks using a mucin emulsifier and its application. Background Technology

[0002] Cytopathology is a method for the pathological diagnosis and research of diseases, offering advantages such as ease of operation, minimal patient discomfort, and high acceptance. Early cytological slide preparation techniques primarily involved cell smears and embedding methods using cell pellets directly after centrifugation (e.g., centrifuge tube sedimentation, frozen cell block preparation, test tube embedding, and slide cell aggregation). However, cell smears are prone to uneven thickness, cell overlap, loose cell distribution, and dark background, leading to diagnostic difficulties. Embedding cells directly after centrifugation, lacking a medium to bind cells together, is extremely ineffective for specimens with low cell counts, and even with specimens containing many cells, smears are prone to detachment during sectioning, resulting in poor staining and affecting pathological diagnostic results. Therefore, with the increasing clinical demand for accurate pathological diagnosis, early cytological slide preparation techniques such as cell smears and embedding cells directly after centrifugation have become bottlenecks restricting the development of pathology and clinical diagnosis.

[0003] In recent years, techniques for preparing cell blocks using agar, egg white, thrombin, and other media have been reported. These media can aggregate and increase cell numbers, but they also have certain limitations. For example, cell blocks prepared with agar are not very effective and the preparation process is complex; cell blocks prepared with egg white may have staining effects due to the color of the egg white itself; and cell blocks prepared with thrombin may affect cell viability. The introduction of these media requires a complete overhaul of the tissue fixation, dehydration, clearing, and paraffin infiltration procedures, which is cumbersome, requires experimentation to determine the optimal conditions, and may also affect the results of subsequent immunohistochemical and other experimental studies. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a method and application for preparing cell blocks using a mucin emulsifier, which is convenient to operate and has a high rate of excellent subsequent section staining.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] A method for preparing cell blocks using a mucin emulsifier includes the following steps:

[0007] Step 1: Place the cytology specimen into a centrifuge tube and centrifuge. Discard the supernatant to obtain the first separation solution.

[0008] Step 2: Add formaldehyde-alcohol solution to the first separation liquid, mix well, centrifuge, discard the supernatant, and retain some precipitate to obtain the second separation liquid.

[0009] Step 3: Add mucin emulsifier to the second separation solution, mix well, centrifuge, discard the supernatant, and obtain a colloidal cell block. Then transfer the cell block at the bottom of the centrifuge tube to the embedding cassette.

[0010] In step one, the cytological specimen is a non-gynecological specimen.

[0011] In step three, the mass ratio of mucin emulsifier to cytological specimen is 1:4.

[0012] In step three, the mucin emulsifier is a mixture of mucin and distilled water, with a mass ratio of mucin to distilled water of 1:10.

[0013] In step one, the first separation liquid is obtained by centrifugation once or multiple times and discarding the supernatant.

[0014] In step one, after the bloody cytology specimen is placed in a centrifuge tube, red blood cell lysis buffer is added and then centrifuged. The supernatant is discarded to obtain the first separation solution.

[0015] In step one, the centrifugation rate is 2400-2600 r / min and the centrifugation time is 4-6 min; in step two, the centrifugation rate is 2400-2600 r / min and the centrifugation time is 4-6 min; in step three, the centrifugation rate is 2400-2600 r / min and the centrifugation time is 4-6 min.

[0016] The application of a cell block prepared by a mucin emulsifier in hematoxylin-eosin staining and immunohistochemical staining, wherein the cell block is a cell block prepared by the above-described method.

[0017] The cell blocks were fixed in formaldehyde, dehydrated in alcohol, cleared in xylene, impregnated with paraffin, embedded, and sectioned to obtain cell block sections.

[0018] The application of the cell block sections in hematoxylin-eosin staining includes the following steps: xylene staining (5 min twice), 100% ethanol staining (2 min twice), 95% ethanol staining (2 min twice), 80% ethanol staining (1 min), distilled water staining (1 min), hematoxylin staining (5 min), distilled water staining (1 min), 1% hydrochloric acid ethanol staining (2 s), distilled water staining (2 s), 1% ammonia staining (5 s), distilled water staining (1 min), 0.5% eosin staining (2 min), distilled water staining (2 s), 80% ethanol staining (2 s), 95% ethanol staining (2 min twice), 100% ethanol staining (2 min twice), xylene staining (3 min three times), and neutral resin staining. The cells were mounted with a gel to obtain hematoxylin and eosin stained cell blocks. The application of these cell blocks in immunohistochemical staining involves the following steps: sectioning and baking the cell blocks, dewaxing to water, antigen retrieval according to the primary antibody requirements, adding 0.3% H2O2 to the sections, incubating, washing with PBS, adding primary antibody, incubating again, washing with PBS, adding secondary antibody, incubating again, washing with PBS, adding DAB chromogenic reagent, counterstaining with hematoxylin, differentiating with hydrochloric acid and ethanol, blueing with ammonia, clearing with xylene, and mounting with neutral resin to obtain immunohistochemically stained cell block sections.

[0019] The beneficial effects of this invention are as follows: This invention provides a method and application for preparing cell blocks using mucin emulsifier. The preparation method is simple and has low production cost. It does not require changing the original tissue paraffin block preparation procedure (fixation, dehydration, clearing and paraffin infiltration, etc.). The prepared cell blocks have high enrichment efficiency, low cell loss, and do not affect cell viability. The sections can meet the effect requirements of hematoxylin and eosin staining and immunohistochemical staining.

[0020] Cell sections prepared with mucin emulsifier exhibit intact appearance, a regular circular structure, moderate porosity, and uniform thickness. These sections show excellent results in hematoxylin-eosin staining and immunohistochemical staining: hematoxylin-eosin staining provides a clear background and sharp contrast, with cells evenly distributed within circular areas, displaying a three-dimensional morphology; the excellent rate of stained sections is 98.94%. Immunohistochemical staining produces a clean background, with specific staining of both structure and cells; the excellent rate of stained sections is 99.30%, demonstrating broad application prospects. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Figure 1 This is an image of cell blocks prepared using the mucin emulsifier of the present invention;

[0023] Figure 2 These are slice images of cell blocks prepared using the mucin emulsifier of the present invention;

[0024] Figure 3 Images of hematoxylin and eosin stained thyroid papillary carcinoma cells prepared with the mucin emulsifier of this invention;

[0025] Figure 4 Images of hematoxylin and eosin-stained mononuclear tissue cell-derived cells prepared using the mucin emulsifier of this invention;

[0026] Figure 5 Immunohistochemical staining images of CK19 thyroid papillary carcinoma cells prepared with the mucin emulsifier of the present invention;

[0027] Figure 6 These are immunohistochemical staining images of most epithelial-like cells of mononuclear tissue cell origin prepared using the mucin emulsifier of the present invention;

[0028] Figure 7 These are images of hematoxylin and eosin staining of pleural effusion (a lung adenocarcinoma metastasis specimen) prepared on agar.

[0029] Figure 8 These are images of immunohistochemical staining of pleural effusion (a lung adenocarcinoma metastasis specimen) prepared with agar. Detailed Implementation

[0030] Unless otherwise specified in this embodiment, all parts are at room temperature and pressure.

[0031] A method for preparing cell blocks using a mucin emulsifier includes the following steps:

[0032] Step 1: Place the non-gynecological specimen into a centrifuge tube and centrifuge. Discard the supernatant to obtain the first separation solution.

[0033] Step 2: Add 5 mL of formaldehyde-alcohol solution to the first separation solution, mix well, centrifuge, discard the supernatant, and retain some precipitate to obtain the second separation solution.

[0034] Step 3: Add mucin emulsifier to the second separation solution, mix well, centrifuge, discard the supernatant, and obtain a colloidal cell block. Then transfer the cell block at the bottom of the centrifuge tube to the embedding cassette.

[0035] In step three, the mass ratio of mucin emulsifier to cytological specimen is 1:4. The mucin emulsifier is a mixture of mucin and distilled water, with a mass ratio of mucin to distilled water of 1:10. The mucin was purchased from Anhui Cool Biotechnology Co., Ltd. In this embodiment, the ratio of mucin to distilled water is 5g:50ml, and the addition amount is 250μL. After multiple experiments, the best results were found when the ratio of mucin to distilled water was 5g:50ml. When the mucin concentration is too low, the cell blocks cannot coagulate and embed; conversely, when the mucin concentration is too high, the cell blocks become too hard and brittle, resulting in poor fusion between the cell blocks and paraffin, with some paraffin becoming loose and floating, which can easily lead to a significant decrease in the quality of the sections during sectioning.

[0036] In addition, in this embodiment, the mucin emulsifier is a self-prepared, non-commercial kit, which does not require the purchase of special equipment or changes to the original tissue block preparation procedure (fixation, dehydration, clearing and paraffin infiltration, etc.). The preparation cost is low, the operation process is simple, and the staining effect is better, which is conducive to its promotion and use in pathology departments of medical units at all levels.

[0037] In step one, the first separation solution is prepared by single or multiple centrifugations followed by discarding the supernatant. If the cell count of non-gynecological specimens is insufficient, the first separation solution can be prepared by repeated centrifugation followed by discarding the supernatant. For example, for specimens with a small cell count and no obvious precipitate visible to the naked eye after the first centrifugation, the rotation speed can be appropriately increased to 3000 rpm, and the centrifugation can be repeated 3 to 5 times until the experimental requirements are met.

[0038] In step one, after the bloody cytology specimen is placed in a centrifuge tube, 30 mL of red blood cell lysis buffer is added and then centrifuged. The supernatant is discarded, and 1 mL of precipitate is retained to obtain the first separation solution. This method is used when the non-gynecological specimen is a bloody specimen.

[0039] In step one, the centrifugation rate is 2400-2600 r / min, and the centrifugation time is 4-6 min; in step two, the centrifugation rate is 2400-2600 r / min, and the centrifugation time is 4-6 min; in step three, the centrifugation rate is 2400-2600 r / min, and the centrifugation time is 4-6 min. The preferred centrifugation rate for all of the above is 2500 r / min (r = 15 cm), and the preferred centrifugation time is 5 min.

[0040] This embodiment provides a method for preparing cell blocks using mucin emulsifier. The preparation method is simple, the production cost is low, the cell block enrichment efficiency is high, the cell loss is small, and the cell viability is not affected. It can meet the effect requirements of hematoxylin-eosin staining and immunohistochemical staining.

[0041] The application of a cell block prepared by a mucin emulsifier in hematoxylin-eosin staining and immunohistochemical staining, wherein the cell block is a cell block prepared by the above-described method.

[0042] The cell blocks were fixed in formaldehyde, dehydrated in alcohol, cleared in xylene, impregnated with paraffin, embedded, and sectioned to obtain cell block sections.

[0043] The application of the cell block sections in hematoxylin-eosin staining involves the following steps: staining the cell block sections twice with xylene for 5 min each time, twice with 100% ethanol for 2 min each time, twice with 95% ethanol for 2 min each time, once with 80% ethanol for 1 min, once with distilled water for 1 min, then staining with hematoxylin for 5 min, once with distilled water for 1 min, then staining with 1% hydrochloric acid ethanol for 2 seconds, once with distilled water for 2 seconds, then staining with 1% ammonia for 5 seconds, once with distilled water for 1 min, then staining with 0.5% eosin for 2 min, once with distilled water for 2 seconds, then staining with 80% ethanol for 2 seconds, twice with 95% ethanol for 2 min each time, twice with 100% ethanol for 2 min each time, then staining with xylene for 3 min each time, followed by mounting with neutral resin to obtain hematoxylin-eosin stained cell block sections.

[0044] The cell blocks are used in immunohistochemical staining. The preparation steps for immunohistochemical staining include sectioning and baking the cell blocks (68℃, 15 min), dewaxing to water, antigen retrieval of the tissue according to the requirements of the primary antibody, adding 0.3% H2O2 to the sections, incubating for 10 min, washing with PBS, adding the primary antibody, incubating for 60 min, washing with PBS, adding the secondary antibody, incubating for 15 min, washing with PBS, adding DAB chromogenic reagent, counterstaining with hematoxylin, differentiating with hydrochloric acid and ethanol, blueing with ammonia, clearing with xylene, and mounting with neutral resin to obtain immunohistochemically stained cell block sections.

[0045] Cell sections prepared with mucin emulsifier exhibit intact appearance, a regular circular structure, moderate porosity, and uniform thickness. These sections show excellent results in hematoxylin-eosin staining and immunohistochemical staining: hematoxylin-eosin staining provides a clear background and sharp contrast, with cells evenly distributed within circular areas, displaying a three-dimensional morphology; the excellent rate of stained sections is 98.94%. Immunohistochemical staining produces a clean background, with specific staining of both structure and cells; the excellent rate of stained sections is 99.30%, demonstrating broad application prospects.

[0046] Furthermore, this embodiment allows for the continuous sectioning of cell blocks, which can be preserved long-term. The sections exhibit high concordance with histological diagnosis and have broad application prospects. Especially for the early diagnosis of diseases where histological specimens are unavailable, cell blocks prepared with mucin emulsifiers are a valuable supplement to histological diagnosis.

[0047] Specific applications of cell block sections prepared with mucin emulsifier in hematoxylin-eosin staining and immunohistochemical staining:

[0048] From July 2018 to January 2022, 1418 cytological samples were collected as the study subjects. Cell blocks were prepared using mucin emulsifier as the matrix material, and paraffin sections and hematoxylin-eosin staining were performed. Two senior pathologists made histological diagnoses on the cell blocks. Of these, 285 samples diagnosed as malignant were resectioned and subjected to 2138 related immunohistochemical staining tests. The patients' ages ranged from 19.86 to 85.61 years, with a mean age of (49.85 ± 18.32) years. The 1418 samples were divided into exfoliative cytology specimens (1183 cases, 83.43%) and fine-needle aspiration cytology specimens (235 cases, 16.57%) according to their source. Exfoliative cytology specimens were categorized as follows: pleural and peritoneal effusion (572 cases, 48.35%), mammary discharge (153 cases, 12.93%), sputum (128 cases, 10.82%), urine (104 cases, 8.79%), bronchoscopic lavage fluid (97 cases, 8.20%), pelvic effusion (75 cases, 6.34%), and pericardial effusion (54 cases, 4.57%). Fine-needle aspiration cytology specimens were categorized as follows: thyroid (127 cases, 54.04%), breast (85 cases, 36.17%), and lymph node (23 cases, 9.79%). Of the 285 samples pathologically diagnosed as malignant, 115 were adenocarcinoma (40.36%), 88 were papillary carcinoma (30.87%), 59 were metastatic carcinoma (20.70%), and 23 were squamous cell carcinoma (8.07%).

[0049] The evaluation criteria for the quality of hematoxylin-eosin staining include: tissue section integrity and number of sections (10 points), section thickness and uniformity (10 points), section integrity (10 points), section flatness (10 points), section purity (10 points), section neatness (10 points), section transparency (10 points), staining clarity (10 points), proper mounting position (10 points), and correct label placement (10 points). Excellent sections: ≥90 points; Good sections: 75–89 points; Basically acceptable sections: 60–74 points; Unacceptable sections: ≤59 points. The percentage of excellent sections = [(Number of excellent sections + Number of good sections) / Total number of sections] x 100%.

[0050] The evaluation criteria for the quality of immunohistochemical staining include: High-quality slides: precise localization, clear background, appropriate staining, and specific staining; Excellent slides: relatively precise localization, relatively clear background, relatively reasonable staining, and a small amount of non-specific staining; Unqualified slides: severe antigen diffusion, unreasonable staining, and a large amount of non-specific staining. The percentage of excellent slides = [(number of high-quality slides + number of excellent slides) / total number of slides] x 100%.

[0051] I. Appearance of cell blocks and sections prepared with mucin emulsifier

[0052] Visual inspection reveals that the success rate of preparing cell blocks for non-gynecological cytology samples using mucin emulsifier is 100%, and the preparation method is simple and low-cost, thus possessing broad application prospects. (Reference) Figure 1 It can be seen that the cell blocks have a round, jelly-like colloidal structure, with cells aggregated into clusters and of moderate firmness. (Refer to...) Figure 2 The cell blocks, after being sliced, have an intact appearance, a regular circular structure, and moderate looseness.

[0053] II. Application of cell block sections prepared with mucin emulsifier in hematoxylin-eosin staining or immunohistochemical staining.

[0054] Reference Figure 3 and Figure 4 As seen in the microscopic images, cell blocks prepared with mucin emulsifier can be sliced ​​continuously and uniformly. Hematoxylin and eosin staining produces a clear background, with vibrant nucleoplasm staining, a clear distinction between red and blue, and distinct contrast. The cell nucleus appears blue, while the cytoplasm, muscle fibers, collagen fibers, and erythrocytes show varying degrees of red. Calcium salts and bacteria may appear blue or purplish-blue. Cells are evenly distributed within circular areas, exhibiting a three-dimensional morphology. Figure 3 It is papillary thyroid carcinoma. The epithelioid cells are arranged in a glandular pattern. The cells are crowded, with large nuclei, thickened and irregular nuclear membranes, pale nuclear chromatin, and visible nuclear grooves. Figure 4 These are mononuclear tissue cell-derived cells, with numerous epithelial-like cells whose cytoplasm is stained red. The cells are relatively large, with vacuoles visible in the cytoplasm, and the nuclei are irregularly shaped, with some nuclei being displaced.

[0055] Reference Figure 5 and Figure 6 As seen in the microscopic images, the paraffin sections of cell blocks prepared with mucin emulsifier were of uniform thickness, with a clean background and specific staining in immunohistochemical staining. This is mainly manifested in two aspects: first, the specific reaction products were distributed in specific locations, indicating structural characteristics; second, the specific products showed varying degrees of positive staining on the same section, indicating cellular characteristics. Figure 5 CK19 showed diffuse strong positivity in the cytoplasm of tumor cells. Figure 6 Most of the epithelioid cells were CD68 positive.

[0056] III. Application of agar-prepared cell block sections in hematoxylin-eosin staining or immunohistochemical staining (comparative example)

[0057] Reference Figure 7Images of hematoxylin and eosin staining of pleural effusion (a lung adenocarcinoma metastasis specimen) prepared on agar are shown. The images show co-staining of the nucleus and cytoplasm, with the nucleus and cytoplasm showing lighter staining and blurred appearance, making the tissue structure unclear. Under the microscope, epithelial-like cells are faintly visible, arranged in glandular clusters, with large, deeply stained nuclei and abundant cytoplasm containing vacuoles. Some nuclei are displaced. Mesothelial cells, a few lymphocytes, and erythrocytes are arranged in sheets in the background. Therefore, compared to this example, the cell blocks prepared on agar show poorer results in hematoxylin and eosin staining.

[0058] Reference Figure 8 The images show immunohistochemical staining of pleural effusion (a lung adenocarcinoma metastasis specimen) prepared with agar. The tissue structure is blurred, the staining shows no clear distribution, and cells and surrounding connective tissue are indistinguishably stained, with double images and faintly visible features. Immunohistochemical TTF-1 analysis shows large nuclei, highly stained cells are positive, while mesothelial cells are negative. Therefore, compared to this example, agar-prepared cell sections are less effective for immunohistochemical staining.

[0059] IV. The excellent rate of cell blocks prepared with mucin emulsifier in hematoxylin-eosin staining or immunohistochemical staining.

[0060] A total of 1418 cell block sections were stained with hematoxylin and eosin, classified as follows: 1142 high-quality sections (80.54%), 261 excellent sections (18.40%), 12 basically acceptable sections (0.85%), and 3 unacceptable sections (0.21%). The excellent rate of hematoxylin and eosin staining was 98.94%. 2138 high-quality immunohistochemical staining sections were also collected, classified as follows: 1729 high-quality sections (80.87%), 394 excellent sections (18.43%), and 15 unacceptable sections (0.70%). The excellent rate of immunohistochemical staining of cell block sections was 99.30%. The above experimental data actually reflects the effect of cell blocks prepared with mucin emulsifier after fixation, dehydration, clearing, paraffin embedding, sectioning, hematoxylin and eosin staining, immunohistochemical staining and mounting. The excellent rate of hematoxylin and eosin staining and immunohistochemical staining of cell block sections is greater than 98%, which meets the pathological diagnosis requirements of tertiary hospitals.

[0061] V. Diagnostic efficacy of cell blocks prepared with mucin emulsifier

[0062] In this embodiment, 294 samples with postoperative pathological diagnosis of cancer were used as positive cases (gold standard), and 1124 samples with non-cancerous pathological diagnoses were used as negative cases (gold standard). The diagnostic sensitivity, specificity, concordance rate, and Youden index of cell blocks prepared with mucin emulsifier are shown in Table 1 below. A comprehensive evaluation suggests that cell blocks prepared with mucin emulsifier have high diagnostic efficacy and broad application prospects, especially for the early diagnosis of diseases where histological specimens are unavailable. Cell blocks prepared with mucin emulsifier are a valuable supplement to histological diagnosis.

[0063]

[0064] Table 1 shows the diagnostic efficacy of cell blocks prepared with mucin emulsifier.

[0065] The above embodiments do not limit the scope of protection of this invention. All equivalent modifications and variations made by those skilled in the art without departing from the overall concept of this invention are still within the scope of this invention.

Claims

1. A method for preparing cell blocks using a mucin emulsifier, characterized in that... Includes the following steps: Step 1: Place the cytology specimen into a centrifuge tube and centrifuge. Discard the supernatant to obtain the first separation solution. Step 2: Add formaldehyde-alcohol solution to the first separation liquid, mix well and centrifuge, discard the supernatant, retain some precipitate to obtain the second separation liquid; Step 3: Add mucin emulsifier to the second separation solution, mix well, centrifuge, discard the supernatant, and obtain a colloidal cell block. Then transfer the cell block at the bottom of the centrifuge tube to the embedding cassette. The mass ratio of the mucin emulsifier to the cytological specimen is 1:4; The mucin emulsifier is a mixture of mucin and distilled water, with a mass ratio of mucin to distilled water of 1:

10.

2. The method for preparing cell blocks using mucin emulsifier according to claim 1, characterized in that... In step one, the cytological specimen is a non-gynecological specimen.

3. The method for preparing cell blocks using a mucin emulsifier according to claim 1 or 2, characterized in that... In step one, the first separation liquid is obtained by centrifugation once or multiple times and discarding the supernatant.

4. The method for preparing cell blocks using a mucin emulsifier according to claim 1 or 2, characterized in that... In step one, after the bloody cytology specimen is placed in a centrifuge tube, red blood cell lysis buffer is added and then centrifuged. The supernatant is discarded to obtain the first separation solution.

5. The method for preparing cell blocks using a mucin emulsifier according to claim 1 or 2, characterized in that... In step one, the centrifugation rate is 2400-2600 r / min and the centrifugation time is 4-6 min; in step two, the centrifugation rate is 2400-2600 r / min and the centrifugation time is 4-6 min; in step three, the centrifugation rate is 2400-2600 r / min and the centrifugation time is 4-6 min.

6. The application of cell blocks prepared with a mucin emulsifier in hematoxylin-eosin staining and immunohistochemical staining, characterized in that... The cell block is the cell block obtained by the method according to any one of claims 1 to 5.

7. The application of cell blocks prepared with the mucin emulsifier according to claim 6 in hematoxylin-eosin staining and immunohistochemical staining, characterized in that... The cell blocks were fixed in formaldehyde, dehydrated in alcohol, cleared in xylene, impregnated with paraffin, embedded, and sectioned to obtain cell block sections.

8. The application of cell blocks prepared with the mucin emulsifier according to claim 7 in hematoxylin-eosin staining and immunohistochemical staining, characterized in that... The application of the cell block sections in hematoxylin-eosin staining includes the following steps: xylene staining (5 min twice), 100% ethanol staining (2 min twice), 95% ethanol staining (2 min twice), 80% ethanol staining (1 min), distilled water staining (1 min), hematoxylin staining (5 min), distilled water staining (1 min), 1% hydrochloric acid ethanol staining (2 s), distilled water staining (2 s), 1% ammonia staining (5 s), distilled water staining (1 min), 0.5% eosin staining (2 min), distilled water staining (2 s), 80% ethanol staining (2 s), 95% ethanol staining (2 min twice), 100% ethanol staining (2 min twice), xylene staining (3 min three times), and neutral resin staining. The cells were mounted with a gel to obtain hematoxylin and eosin stained cell blocks. The application of these cell blocks in immunohistochemical staining involves the following steps: sectioning and baking the cell blocks, dewaxing to water, antigen retrieval according to the primary antibody requirements, adding 0.3% H2O2 to the sections, incubating, washing with PBS, adding primary antibody, incubating again, washing with PBS, adding secondary antibody, incubating again, washing with PBS, adding DAB chromogenic reagent, counterstaining with hematoxylin, differentiating with hydrochloric acid and ethanol, blueing with ammonia, clearing with xylene, and mounting with neutral resin to obtain immunohistochemically stained cell block sections.

Images