Cytopathological staining kit and staining method

Abstract

Provided in the present invention are a cytopathological staining kit and a staining method. The staining kit comprises a basic fuchsin staining reagent, a silver nitrate staining reagent, an ethanol-

Description

The present application claims the priority benefit of Chinese application No. 202410762100.1, filed on June 13, 2024, entitled "Cytopathological Staining Kit and Staining Method", the entirety of which is hereby incorporated by reference. TECHNICAL FIELD The present application belongs to the field of pathological examination and analysis, and particularly relates to a cytopathological staining kit and a staining method. BACKGROUND ART A human cell consists of three parts: cell membrane, cell slurry (cytoplasm), and cell nucleus. However, cytopathology should be particularly focuses on structure and morphology of the cell nucleus and the nucleolus in the cell nucleus. The nucleolus in the cell nucleus is a crucial organelle and the hub of all cellular activities. Changes in the cell nucleus, especially changes in its nucleolus, are the basis and core evidence for identifying the pathological structure of and morphology of malignant cells. The nucleolus contains nucleolar markers such as fibrillarin, nucleophosmin, nucleolin, RNA polymerase I, DNA topoisomerase, upstream binding factor, and rRNA and rDNA. There are significant differences in the morphological structure of the cell nucleus and nucleolus between normal cells and malignant cells. Currently, there are generally four staining methods or technologies for detecting malignant cells or pathological tissues, that is, histopathological examination, bone marrow cytological examination, exfoliative cytological examination, and liquid biopsy of circulating tumor cells (CTCs) in peripheral blood. The histopathological examination mainly uses HE and histochemical staining to detect and identify malignant cells, and can only visualize the basic structures of cells, such as the morphological structures of the cell membrane, cytoplasm, and cell nucleus. Its main technical drawback is that it cannot clearly display the morphology of the nucleolus, cannot specifically label the nucleolus, and cannot distinguish the nucleolus from nucleoplasm. The result of chemical staining used in the exfoliative cytological examination is basically the same as that of the histopathological examination. It can reveal the basic structures of cells and the cell nucleus, but cannot clearly display the morphology and structure of the nucleolus, cannot label the nucleolus, and cannot distinguish the nucleolus from nucleoplasm. The bone marrow cytological examination adopts chemical staining, which can reveal the overall cellular structure and shows the cell nucleus and nucleolus. However, the bone marrow cytology adopts a routine chemical staining, which has low sensitivity for nucleolus staining and cannot distinguish the nucleolus from nucleoplasm, bringing uncertainty to the cytological diagnosis of hematological diseases. The chemical staining currently used in CTC cytology testing has poor repeatability and low sensitivity, and basically cannot clearly visualize the morphology and structure of the nucleolus. The mainstream immunofluorescence staining method focuses on displaying antigens on the cell surface, but cannot show the morphology and structure of the nucleolus in a cell nucleus. It is determined whether a cell is a CTC cell by the stained nucleus with antigens or DAPI, and false positives or false negatives can occur due to changes in antigens or illegal editing of antigens. CTCs cannot be examined and identified from the cytopathological characteristics. SUMMARY OF THE INVENTION To address some of the shortcomings in the prior art, the present application provides a cytopathological staining kit and a staining method, which can clearly show the cell membrane, cytoplasm, and cell nucleus, especially the morphology of the nucleolus in the cell nucleus. A first aspect of the present application provides a cytopathological staining kit, comprising a basic fuchsin staining reagent, a silver nitrate staining reagent, an ethanol-hydrochloric acid fixative, a gelatin-formic acid reducing solution, and a washing solution. In one embodiment, the basic fuchsin staining reagent is prepared from basic fuchsin, acetic acid, and water. In one embodiment, the basic fuchsin staining reagent is prepared from basic fuchsin, acetic acid, and water in a ratio of 0.1(g): l(mL): 100(mL). In one embodiment, the silver nitrate staining reagent is an aqueous silver nitrate solution with a concentration of l-3mol / L. In one embodiment, the ethanol-hydrochloric acid fixative is prepared from ethanol and hydrochloric acid, and a concentration of the hydrochloric acid in the ethanol-hydrochloric acid fixative is 0.5-2mol / L. In one embodiment, the ethanol-hydrochloric acid fixative is prepared from ethanol and concentrated hydrochloric acid, and a concentration of hydrochloric acid in the ethanolhydrochloric acid fixative is 0.5-2mol / L. In one embodiment, the gelatin-formic acid reducing solution is prepared from gelatin, formic acid, and water; in the gelatin-formic acid reducing solution, a mass percentage of the gelatin is l%-5%, and a mass percentage of the formic acid is 0.5-4%. In one embodiment, a volume ratio of the silver nitrate staining reagent to the gelatin-formic acid reducing solution is (1.5-2.1): (0.9-1.2). In one embodiment, the washing solution is PBS (Phosphate Buffered Saline) containing Tween-20 with a pH of 7.2-7.4. A second aspect of the present application provides a cytopathological staining method, adopting the cytopathological staining kit described in any one of the foregoing embodiments, comprising the following steps: (1) preparing a human tissue cell specimen slide; (2) fixing cells on the specimen slide with the ethanol-hydrochloric acid fixative to expose aldehyde groups of nuclear DNA, then rinsing with the washing solution after treatment; (3) mixing the silver nitrate staining reagent with the gelatin-formic acid reducing solution to obtain a mixed reagent; adding the mixed reagent, then rinsing with the washing solution after treatment; (4) adding the basic fuchsin staining reagent, then rinsing with the washing solution after treatment; (5) air-drying, mounting with a mounting medium, and examining under an optical microscope. More specifically, the staining method comprises: (1) preparing or making the human tissue cell specimen slide; (2) fixing the cells on the specimen slide with the ethanol-hydrochloric acid fixative to expose aldehyde groups of the nuclear DNA, treating at 37°C for 10-15 minutes, and rinsing three times with the washing solution; (3) mixing the silver nitrate staining reagent with the gelatin-formic acid reducing solution in a volume ratio of (1.5-2.1):(0.9-1.2) to obtain the mixed reagent; adding the mixed reagent, staining at 37°C for 12-18 minutes, and rinsing three times with the washing solution; (4) adding the basic fuchsin staining reagent, staining at 37°C for 12-18 minutes, and rinsing three times with the washing solution; (5) air-drying, mounting with the mounting medium, and examining under the optical microscope. A third aspect of the present application provides use of the cytopathological staining kit described in any one of the foregoing embodiments, which is capable of staining the cell membrane, cytoplasm, and nucleus of cells to show morphology of the cell membrane, cytoplasm, and nucleus. In one embodiment, morphology of nucleolus of the cell nucleus is able to be further showed. As is well known to those skilled in the art, the morphology of the cell nucleus mainly refers to the shape, size, structure, and dynamic changes; the morphology of the nucleoli mainly refers to the shape, size, quantity, structural partitioning, and dynamic changes. In the present application, silver nitrate and basic fuchsin staining can not only show the staining differences between the cytoplasm and nucleus, but also can show the morphology of the nucleolus in the cell nucleus distinctly and completely. A fourth aspect of the present application provides use of the cytopathological staining kit described in any one of the foregoing embodiments, which is used for staining and identifying normal cells and pathological cells. Compared with the prior art, the beneficial effects of the present application are as follows: (1) The cytopathological staining kit and the staining method provided in at least one embodiment of the present application can display a complete cell structure: the silver nitrate staining reagent is combined with the basic fuchsin staining reagent, and after staining, the cell clearly shows the whole cell structure of the cell membrane, the cell slurry, the nucleus, and the nucleolus; in particular, the morphology of the nucleolus of the nucleus is displayed, including features such as shape, quantity, size, and distribution. (2) The cytopathological staining kit and the staining method provided in at least one embodiment of the present application can display the structure and morphology of the nucleolus in the nucleus: nucleolar markers (substances in the nucleolus) in the nucleus, such as fibrillarin, nucleophosmin, nucleolin, RNA polymerase I, DNA topoisomerase, upstream binding factor, and rRNAand rDNA etc. are stained by the silver nitrate staining reagent, which can directly display and mark the nucleolus, showing and determining the morphology of the nucleolus. (3) The cytopathological staining kit and the staining method provided in at least one embodiment of the present application enable the quantitative staining of DNA in the nucleus: the DNA content in the nucleus is closely related to the cell nucleus size, the chromatin texture, and the nucleus staining depth. The ethanol-hydrochloric acid fixative in the present kit can make the nuclear DNA depurine exposed aldehyde group more likely to be stained red or purple-red by the basic fuchsin, show the chromatin texture, and the red or purple-red depth is directly proportional to the intracellular DNA content, so it can be extended for the nuclear DNA content assay (DNA ploidy analysis). (4) The cytopathological staining kit and the staining method provided in at least one embodiment of the present application can display a clear and complete cell structure, in particular to display nucleus, especially the nucleolus capable of be clearly identified with its morphology clearly visible. (5) The cytopathological staining kit and the staining method provided in at least one embodiment of the present application are widely used, which can be applied to pathological staining and detection of blood cytopathological cells (including CTCs and blood disease cells), and can also be applied to pathological staining and detection of exfoliated cells (including pleural effusion, ascites, cerebrospinal fluid, bronchoalveolar lavage fluid, cervical scraper, etc.), and can also be applied to pathological tissue cell biopsy (including section pathological tissue cell biopsy and fine needle biopsy); In addition, the staining results of the present application can also be extended for nucleus DNA content assays (DNA ploidy analysis). BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1-4 are optical microscope photographs of the staining results of Examples 1-4, respectively; FIGs. 5-8 are optical microscope photographs of the staining results of Comparative Examples 1-4, respectively; FIGs. 9-12 are optical microscope photographs of the staining results of Comparative Examples 5-8, respectively; FIGs. 13-16 are optical microscope photographs of the staining results of Comparative Examples 9-12, respectively; FIGs. 17-20 are optical microscope photographs of the staining results of Comparative Examples 13-16, respectively; FIGs. 21-23 are optical microscope photographs of the staining results of Examples 5-7, respectively; FIGs. 24-26 are optical microscope photographs of the staining results of Comparative Examples 17-19, respectively; FIGs. 27-29 are optical microscope photographs of the staining results of Examples 8-10, respectively. DETAILED DESCRIPTION OF THE EMBODIMENTS The present application is described in detail below with reference to examples, and it should be understood that these examples are merely some preferred examples of the present application, and cannot be understood as limiting of the protection scope of the present application. The reagents used in the examples of the present application specifically comprises the following components: A basic fuchsin staining reagent, prepared from basic fuchsin (chemical formula: C19H18CIN3), acetic acid, and water in a ratio of 0.1g: 1mL: 100mL. A silver nitrate staining reagent, being an aqueous silver nitrate solution with a concentration of 1 -3 mol / L (e.g., 1.5 mol / L, 2 mol / L, 2.5 mol / L, etc.), and added with 0.2-3.0 wt% dimethyl sulfoxide (DMSO), i.e. a mass percentage of the DMSO in the silver nitrate staining reagent is 0.2-3.0%. An ethanol-hydrochloric acid fixative, prepared by from ethanol and concentrated hydrochloric acid; wherein, a concentration of the hydrochloric acid in the ethanol-hydrochloric acid fixative is 0.5-2 mol / L, for example, 1 mol / L, 1.5 mol / L, etc. A gelatin-formic acid reducing solution, prepared from gelatin, formic acid, and water; wherein, a mass concentration of the gelatin is l%-5%, i.e., the mass percentage of the gelatin in the gelatin-formic acid reducing solution is l%-5% (e.g., 2%, 3%, 4%, etc.); and a mass concentration of the formic acid is 0.5%-4%, i.e., the mass percentage of the formic acid in the gelatin-formic acid reducing solution is 0.5-4%, for example, 1%, 2%, 3%, etc. A washing solution, being PBS (Phosphate Buffered Saline) containing Tween-20 with a pH of 7.2-7.4. A Schiff’s reagent, prepared from basic fuchsin, water, hydrochloric acid, and sodium bisulfite in a ratio of 0.5 g:100 mL:10 mL:l g, wherein, a concentration of the hydrochloric acid is 1.5 mol / L. A HE reagent, consisting a mixture of hematoxylin staining reagent and eosin staining reagent. The hematoxylin staining reagent is prepared from hematoxylin, ethanol, alum, water, mercuric oxide, and acetic acid in a ratio of 2.5 g:25 mL:2.5 g:500 mL:1.25 g:20 mL. The eosin staining reagent is prepared from eosin, water, ethanol, and acetic acid in a ratio of 1 g:75 mL:25 mL: 1 mL. Example 1: Cytopathological staining and detection of CTCs: (1) A CTC staining and detection slide was prepared; (2) Cells were fixed by the ethanol-hydrochloric acid fixative to expose aldehyde groups of the nucleus DNA, treated at 37°C for 10 minutes, then rinsed three times with the washing solution; wherein the concentration of the hydrochloric acid in the ethanol-hydrochloric acid fixative was 1.5 mol / L; (3) The silver nitrate staining reagent was mixed with the gelatin-formic acid reducing solution in a volume ratio of 1.5:1.0, and the mixed reagent was added, stained at 37°C for 15 minutes, and rinsed three times with the washing solution; wherein the concentration of silver nitrate in the silver nitrate staining reagent was 1 mol / L, containing 0.2 wt% dimethyl sulfoxide; the mass percentage of gelatin in the gelatin-formic acid reducing solution was 1%, and the mass percentage of formic acid was 2%; (4) The basic fuchsin staining reagent was added for staining at 37°C for 15 minutes, and then rinsed three times with the washing solution; (5) Air-drying, mounting with a mounting medium and examination under an optical microscope were performed. Example 2: Cytopathological staining and detection of CTCs: (1) A CTC staining and detection slide was prepared; (2) Cells were fixed with the ethanol-hydrochloric acid fixative to expose aldehyde groups of the nucleus DNA, treated at 37°C for 10 minutes, then rinsed three times with the washing solution; wherein the concentration of hydrochloric acid in the ethanol-hydrochloric acid fixative was 1.5 mol / L; (3) The silver nitrate staining reagent was mixed with the gelatin-formic acid reducing solution in a volume ratio of 2.0:1.0, and the mixed reagent was added, stained at 37°C for 15 minutes, and rinsed three times with the washing solution; wherein the concentration of silver nitrate in the silver nitrate staining reagent was 1 mol / L, containing 0.2 wt% dimethyl sulfoxide; the mass percentage of gelatin in the gelatin-formic acid reducing solution was 2%, and the mass percentage of formic acid was 2%; (4) The basic fuchsin staining reagent was added for staining at 37°C for 15 minutes, and then rinsed three times with the washing solution; (5) Air-drying, mounting with a mounting medium and examination under an optical microscope were performed. Example 3: Cytopathological staining and detection of CTCs: (1) A CTC staining and detection slide was prepared; (2) Cells were fixed with the ethanol-hydrochloric acid fixative to expose aldehyde groups of the nucleus DNA, treated at 37°C for 15 minutes, then rinsed three times with the washing solution; wherein the concentration of hydrochloric acid in the ethanol-hydrochloric acid fixative was 1.5 mol / L; (3) The silver nitrate staining reagent was mixed with the gelatin-formic acid reducing solution in a volume ratio of 2.1:1.0, and the mixed reagent was added, stained at 37°C for 15 minutes, and rinsed three times with the washing solution; wherein the concentration of silver nitrate in the silver nitrate staining reagent was 1 mol / L, containing 0.2 wt% dimethyl sulfoxide; the mass percentage of gelatin in the gelatin-formic acid reducing solution was 5%, and the mass percentage of formic acid was 4%; (4) The basic fuchsin staining reagent was added for staining at 37°C for 18 minutes, and then rinsed three times with the washing solution; (5) Air-drying, mounting with a mounting medium and examination under an optical microscope were performed. Example 4: Cytopathological staining and detection of CTCs: (1) A CTC staining and detection slide was prepared; (2) Cells were fixed with the ethanol-hydrochloric acid fixative to expose aldehyde groups of the nucleus DNA, treated at 3 7°C for 10-15 minutes, then rinsed three times with the washing solution; wherein the concentration of hydrochloric acid in the ethanol-hydrochloric acid fixative was 1.5 mol / L; (3) The silver nitrate staining reagent was mixed with the gelatin-formic acid reducing solution in a volume ratio of 1.5:0.9, and the mixed reagent was added, stained at 37°C for 12 minutes, and rinsed three times with the washing solution; wherein the concentration of silver nitrate in the silver nitrate staining reagent was 1 mol / L, containing 0.2 wt% dimethyl sulfoxide; the mass percentage of gelatin in the gelatin-formic acid reducing solution was 3%, and the mass percentage of formic acid was 3%; (4) The basic fuchsin staining reagent was added for staining at 37°C for 12 minutes, and then rinsed three times with the washing solution; (5) Air-drying, mounting with a mounting medium and examination under an optical microscope were performed. Optical microscope photographs of the staining results of Examples 1-4 are presented in FIGs. 1-4, respectively; among them, the cell membrane and cytoplasm appear light pale red; the cell nucleus appears dark red or purplish red, with visible chromatin texture; the nucleolus of the cell nucleus appears brownish black, displaying the quantity, size, morphology, and distribution of the nucleolus. Comparative Example 1: The step (4) of Example 1 was omitted; other steps were the same as those in Example 1. Comparative Example 2: The step (4) of Example 2 was omitted; other steps were the same as those in Example 2. Comparative Example 3: The step (4) of Example 3 was omitted; other steps were the same as those in Example 3. Comparative Example 4: The step (4) of Example 4 was omitted; other steps were the same as those in Example 4. The staining results of Comparative Examples 1 to 4 are presented in FIGs. 5-8, respectively. Comparative Example 5: The step (3) of Example 1 was omitted; other steps were the same as those in Example 1. Comparative Example 6: The step (3) of Example 2 was omitted; other steps were the same as those in Example 2. Comparative Example 7: The step (3) of Example 3 was omitted; other steps were the same as those in Example 3. Comparative Example 8: The step (3) of Example 4 was omitted; other steps were the same as those in Example 4. Optical microscope photographs of the staining results of Comparative Examples 5-8 are presented in FIGs. 9-12, respectively. Comparative Example 9: The basic fuchsin staining reagent in the step (4) of Example 1 was replaced by the Schiffs reagent; other steps were the same as those in Example 1. Comparative Example 10: The basic fuchsin staining reagent in the step (4) of Example 2 was replaced by the Schiffs reagent; other steps were the same as those in Example 2. Comparative Example 11: The basic fuchsin staining reagent in the step (4) of Example 3 was replaced by the Schiffs reagent; other steps were the same as those in Example 3. Comparative Example 12: The basic fuchsin staining reagent in the step (4) of Example 4 was replaced by the Schiffs reagent; other steps were the same as those in Example 4. Optical microscope photographs of the staining results of Comparative Examples 9-12 are presented in FIGs. 13-16, respectively. Comparative Example 13: The step (3) of Example 1 was omitted; the basic fuchsin staining reagent in the step (4) of Example 1 was replaced by the HE reagent; other steps were the same as those in Example 1. Comparative Example 14: The step (3) of Example 2 was omitted; the basic fuchsin staining reagent in the step (4) of Example 2 was replaced by the HE reagent; other steps were the same as those in Example 2. Comparative Example 15: The step (3) of Example 3 was omitted; the basic fuchsin staining reagent in the step (4) of Example 3 was replaced by the HE reagent; other steps were the same as those in Example 3. Comparative Example 16: The step (3) of Example 4 was omitted; the basic fuchsin staining reagent in the step (4) of Example 4 was replaced by the HE reagent; other steps were the same as those in Example 4. Optical microscope photographs of the staining results of Comparative Examples 13-16 are presented in FIGs. 17-20, respectively. By comparing the photographs of the staining results of the above Examples 1-4 (FIGs. 1-4) with those of Comparative Examples 1-16 (FIGs. 5-20), it can be seen that the overall staining photographs of cells stained using the silver nitrate-basic fuchsin staining method of the present application are clear; the cell membrane, cytoplasm, cell nucleus, and nucleoli of the cell nucleus are intact with clear boundaries. The cytoplasm appears light pale red, the cell nucleus appears red, the nucleoli appear brownish black spots; the morphology of the nucleoli, including quantity and size, is clearly visible. In contrast, single staining with silver nitrate reagent, single staining with basic fuchsin reagent, staining with silver nitrate-Schiff s reagent, and staining with HE reagent all exhibit poorer discriminability of cell components compared with the silver nitrate-basic fuchsin reagent staining of the present application. Example 5: Biopsy of pathological tissue cells: (1) A pathological tissue cell detection slide was prepared; (2) Cells were fixed with the ethanol-hydrochloric acid fixative to expose aldehyde groups of the nucleus DNA, treated at 37°C for 10 minutes, then rinsed three times with the washing solution; wherein the concentration of hydrochloric acid in the ethanol-hydrochloric acid fixative was 1.5 mol / L; (3) The silver nitrate staining reagent was mixed with the gelatin-formic acid reducing solution in a volume ratio of 1.5:1.0, and the mixed reagent was added, stained at 37°C for 15 minutes, and rinsed three times with the washing solution; wherein the concentration of silver nitrate in the silver nitrate staining reagent was 1 mol / L, containing 0.2 wt% dimethyl sulfoxide; the mass percentage of gelatin in the gelatin-formic acid reducing solution was 1%, and the mass percentage of formic acid was 2%; (4) The basic fuchsin staining reagent was added for staining at 37°C for 15 minutes, and then rinsed three times with the washing solution; (5) Air-drying, mounting with a mounting medium and examination under an optical microscope were performed. Example 6: Biopsy of pathological tissue cells: (1) A pathological tissue cell detection slide was prepared; (2) Cells were fixed with the ethanol-hydrochloric acid fixative to expose aldehyde groups of the nucleus DNA, treated at 37°C for 10 minutes, then rinsed three times with the washing solution; wherein the concentration of hydrochloric acid in the ethanol-hydrochloric acid fixative was 1.5 mol / L; (3) The silver nitrate staining reagent was mixed with the gelatin-formic acid reducing solution in a volume ratio of 2.0:1.0, and the mixed reagent was added, stained at 37°C for 15 minutes, and rinsed three times with the washing solution; wherein the concentration of silver nitrate in the silver nitrate staining reagent was 1 mol / L, containing 0.2 wt% dimethyl sulfoxide; the mass percentage of gelatin in the gelatin-formic acid reducing solution was 2%, and the mass percentage of formic acid was 2%; (4) The basic fuchsin staining reagent was added for staining at 37°C for 15 minutes, and then rinsed three times with the washing solution; (5) Air-drying, mounting with a mounting medium and examination under an optical microscope were performed. Example 7: Biopsy of pathological tissue cells: (1) A pathological tissue cell detection slide was prepared; (2) Cells were fixed with the ethanol-hydrochloric acid fixative to expose aldehyde groups of the nucleus DNA, treated at 37°C for 15 minutes, then rinsed three times with the washing solution; wherein the concentration of hydrochloric acid in the ethanol-hydrochloric acid fixative was 1.5 mol / L; (3) The silver nitrate staining reagent was mixed with the gelatin-formic acid reducing solution in a volume ratio of 2.1:1.0, and the mixed reagent was added, stained at 37°C for 15 minutes, and rinsed three times with the washing solution; wherein the concentration of silver nitrate in the silver nitrate staining reagent was 1 mol / L, containing 0.2 wt% dimethyl sulfoxide; the mass percentage of gelatin in the gelatin-formic acid reducing solution was 5%, and the mass percentage of formic acid was 4%; (4) The basic fuchsin staining reagent was added for staining at 37°C for 18 minutes, and then rinsed three times with the washing solution; (5) Air-drying, mounting with a mounting medium and examination under an optical microscope were performed. Comparative Example 17: The step (3) of Example 5 was omitted; the basic fuchsin staining reagent in the step (4) of Example 5 was replaced by the HE reagent; other steps were the same as those in Example 5. Comparative Example 18: The step (3) of Example 6 was omitted; the basic fuchsin staining reagent in the step (4) of Example 6 was replaced by the HE reagent; other steps were the same as those in Example 6. Comparative Example 19: The step (3) of Example 7 was omitted; the basic fuchsin staining reagent in the step (4) of Example 7 was replaced by the HE reagent; other steps were the same as those in Example 7. Optical microscope photographs of the staining results of Examples 5-7 are shown in FIGs. 21-23, respectively. Optical microscope photographs of the staining results of Examples 17-19 are shown in FIGs. 24-26, respectively. By comparing the six photographs of the staining results of paraffin sections of pathological tissues, it can be seen that HE staining can only display the photographs of cell nucleus, but the photographs of the nucleoli are quite blurred with low resolution; however, the staining method of the present application can not only clearly display the intact cell nucleus, but also clearly display the morphology of nucleoli, including their quantity, size, and distribution, and the like. Example 8: Cytopathological staining and detection of CTCs: (1) A CTC staining and detection slide was prepared; (2) Cells were fixed with the ethanol-hydrochloric acid fixative to expose aldehyde groups of the nucleus DNA, treated at 37°C for 10 minutes, then rinsed three times with the washing solution; wherein the concentration of hydrochloric acid in the ethanol-hydrochloric acid fixative was 0.5 mol / L; (3) The silver nitrate staining reagent was mixed with the gelatin-formic acid reducing solution in a volume ratio of 1.5:1.0, and the mixed reagent was added, stained at 37°C for 15 minutes, and rinsed three times with the washing solution; wherein the concentration of silver nitrate in the silver nitrate staining reagent was 1 mol / L, containing 0.2 wt% dimethyl sulfoxide; the mass percentage of gelatin in the gelatin-formic acid reducing solution was 1%, and the mass percentage of formic acid was 2%; (4) The basic fuchsin staining reagent was added for staining at 37°C for 15 minutes, and then rinsed three times with the washing solution; (5) Air-drying, mounting with a mounting medium and examination under an optical microscope were performed. Example 9: The concentration of hydrochloric acid in the ethanol-hydrochloric acid fixative in the step (2) of Example 8 was changed to 1.5 mol / L; other steps were the same as those in Example 8. Example 10: The concentration of hydrochloric acid in the ethanol-hydrochloric acid fixative in the step (2) of Example 8 was changed to 2.0 mol / L; other steps were the same as those in Example 8. Optical microscope photographs of the staining results of Examples 8-10 are shown in FIGs. 27-29, respectively. By comparing these photographs, it can be found that the ethanol-hydrochloric acid fixative with a hydrochloric acid concentration of 1.5 mol / L has the best staining effect. The examples are only described as preferred examples of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements made on the technical solutions of the present application by ordinary skill in the art without departing from the design spirit of the present application shall fall within the protective scope confirmed by the claims of the present application.

Claims

1. A cytopathological staining kit, wherein, comprising a basic fuchsin staining reagent, a silver nitrate staining reagent, an ethanol-hydrochloric acid fixative, a gelatin-formic acid reducing solution, and a washing solution.

2. The cytopathological staining kit according to claim 1, wherein, the basic fuchsin staining reagent is prepared from basic fuchsin, acetic acid, and water.

3. The cytopathological staining kit according to claim 1, wherein, the basic fuchsin staining reagent is prepared from basic fuchsin, acetic acid, and water in a ratio of 0.1(g): 1 (mL): 100(mL).

4. The cytopathological staining kit according to claim 1, wherein, the silver nitrate staining reagent is an aqueous silver nitrate solution with a concentration of l-3mol / L.

5. The cytopathological staining kit according to claim 1, wherein, the ethanol-hydrochloric acid fixative is prepared from ethanol and hydrochloric acid, and a concentration of the hydrochloric acid in the ethanol-hydrochloric acid fixative is 0.5-2mol / L.

6. The cytopathological staining kit according to claim 1, wherein, the gelatin-formic acid reducing solution is prepared from gelatin, formic acid, and water; in the gelatin-formic acid reducing solution, a mass percentage of the gelatin is l%-5%, and a mass percentage of the formic acid is 0.5-4%.

7. The cytopathological staining kit according to claim 1, wherein, a volume ratio of the silver nitrate staining reagent to the gelatin-formic acid reducing solution is (1.5-2.1): (0.9-1.2).

8. The cytopathological staining kit according to claim 1, wherein, the washing solution is Phosphate Buffered Saline (PBS) containing Tween-20 with a pH of 7.2-7.4.

9. A cytopathological staining method, wherein, adopting the cytopathological staining kit according to any one of claims 1-8, and comprising the following steps:(1) preparing a human tissue cell specimen slide;(2) fixing cells on the specimen slide with the ethanol-hydrochloric acid fixative to expose aldehyde groups of nuclear DNA, then rinsing with the washing solution after treatment;(3) mixing the silver nitrate staining reagent with the gelatin-formic acid reducing solution to obtain a mixed reagent; adding the mixed reagent, then rinsing with the washing solution after treatment;(4) adding the basic fuchsin staining reagent, then rinsing with the washing solution after treatment;(5) air-drying, mounting with a mounting medium, and examining under an optical microscope.

10. Use of the cytopathological staining kit according to any one of claims 1-8, being able to stain cell membrane, cytoplasm, and nucleus of a cell, and being able to show morphology of nucleolus of the nucleus.

11. Use of the cytopathological staining kit according to any one of claims 1-8, for staining and identifying normal cells and pathological cells.

Images