A cytopathology staining kit and staining method

Abstract

The invention discloses a cytopathology staining kit and a staining method, wherein the staining kit comprises 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. Cells are fixed with an ethanol hydrochloric acid solution to expose the aldehyde groups of the cell nucleus DNA, and the cells are jointly stained with silver nitrate and basic fuchsin to reveal the complete cell structure and morphology. In particular, the cell nucleolus can be clearly and completely displayed, especially the nucleolus morphology, number, volume and distribution of the cell nucleus can be calibrated, and it can also be extended to be used for the determination of the cell nuclear DNA content.

Description

Technical Field

[0001] The present invention generally relates to the field of pathological testing and analysis, and in particular to a cytopathology staining kit and a staining method. Background Art

[0002] Human cells are composed of three parts: the cell membrane, cytoplasm, and nucleus. However, cytopathology particularly focuses on the nucleus of pathological cells and the structure and morphology of the nucleolus within the nucleus. The nucleolus is a crucial organelle within the nucleus, the hub of all cellular activity. Changes in the nucleus, particularly those within the nucleolus, are crucial and provide the foundation and core evidence for identifying the pathological structure and morphology of malignant cells.

[0003] The nucleolus in the cell nucleus is structured into three parts: the fibrous center, the dense fibrous component, and the granular component. The fibrous center is the innermost structure of the nucleolus, accounting for 5% of the total nucleolus volume. The dense fibrous component accounts for approximately 15% of the total nucleolus volume. It is the first component to form during nucleolus formation, closely connected to and surrounding the fibrous center. The granular component surrounds the fibrous component and accounts for approximately 75% of the nucleolus. The nucleolus contains nucleolar markers such as nucleolar fibrillarin, nucleolar phosphatase, nucleolin, RNA polymerase I, DNA topoisomerase, upstream binding factors, and rRNA and rDNA. The size and morphology of the nucleus, as well as the size, morphology, number, and distribution of the nucleoli, are highly correlated with the structure and morphology of normal cells and malignant pathological cells.

[0004] Currently, the staining methods or techniques for detecting malignant cells or pathological tissues are roughly divided into four types: histopathological examination, bone marrow cytology examination, exfoliated cell examination, and liquid biopsy of circulating tumor cells (CTCs) in peripheral blood.

[0005] Histopathological examination primarily uses HE and histochemical staining to detect and identify malignant cells. These stains only reveal basic cellular structures, such as the cell membrane, cytoplasm, and nucleus. Their main technical drawback is that they cannot clearly visualize the morphology and structure of the nucleolus, clearly delineate the nucleolus, or distinguish between the nucleolus and the nucleoplasm. Chemical staining methods used for exfoliated cells are essentially the same as histological examination methods. These stains can reveal basic cellular structures and the nucleus, but cannot clearly visualize the morphology and structure of the nucleolus, delineate the nucleolus, or distinguish between the nucleolus and the nucleoplasm.

[0006] Bone marrow cytology uses chemical staining to visualize the overall structure of cells, showing the nucleus and nucleolus. However, bone marrow cytology uses general chemical staining, which has low sensitivity to nucleolus coloring and cannot distinguish between nucleolus and nucleoplasm, which brings certain uncertainty to the cytological diagnosis of blood diseases.

[0007] Current chemical staining methods for CTC cytological testing have poor reproducibility and low sensitivity, and are rarely able to clearly visualize the morphology and structure of the nucleus. Mainstream immunofluorescence staining methods focus on visualizing cell surface antigens but fail to reveal the morphology and structure of the nucleus. CTC identification is determined by antigen or DAPI-stained nuclei, but false positives or negatives can occur due to antigen changes or illicit antigen editing. Consequently, CTCs cannot be detected and identified based on cytopathological features. Summary of the Invention

[0008] The present invention aims to provide a cytopathology staining kit and method. This method not only stains marker proteins within the nucleolus, but also stains the cell membrane, cytoplasm, and nucleus. This method clearly and completely displays the morphology, size, number, and distribution of the cell membrane, cytoplasm, and nucleus, particularly the nucleoli. This method can be used to stain and identify normal and pathological cells.

[0009] According to a first aspect of the present invention, a cytopathology staining kit is provided, 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.

[0010] Specifically, 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).

[0011] Specifically, the silver nitrate staining reagent is a silver nitrate aqueous solution with a concentration of 1-3 mol / L.

[0012] Specifically, the ethanol-hydrochloric acid fixative is prepared from ethanol and concentrated hydrochloric acid, wherein the concentration of the hydrochloric acid is 0.5-2 mol / L.

[0013] Specifically, the gelatin formic acid reduction solution is prepared from gelatin, formic acid and water, wherein the mass concentration of gelatin is 1%-5%, and the mass concentration of formic acid is 0.5-4%.

[0014] Specifically, the washing solution is PBS with a pH of 7.2-7.4 containing Tween-20.

[0015] According to a second aspect of the present invention, a cytopathology staining method is provided, using the cytopathology staining kit described in the first aspect of the present invention, comprising the following steps:

[0016] (1) Prepare or prepare human tissue cell specimens;

[0017] (2) Fix the cells with ethanol-hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treat at 370°C for 10-15 minutes, and rinse three times with washing solution;

[0018] (3) Mix the silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 1.5-2.1:0.9-1.2; add the mixed reagent, stain at 370°C for 12-18 minutes, and rinse with washing solution three times;

[0019] (4) Add basic fuchsin staining reagent and stain at 370°C for 12-18 minutes;

[0020] (5) Rinse with washing solution 3 times;

[0021] (6) Allow to dry, seal with sealing tablets, and examine under an optical microscope.

[0022] The basic principles and key technologies of the present invention are as follows:

[0023] (1) Complete cell structure: Silver nitrate staining reagent is combined with basic fuchsin staining reagent to clearly show the complete cell structure of cell membrane, cytoplasm, nucleus and nucleolus after staining; especially the morphology, number, size and distribution of cell nucleoli.

[0024] (2) Nucleolar structure and morphology in the cell nucleus: Nucleolar markers (substances within the nucleus) such as nucleolar fibrillin, nucleolar phosphoprotein, nucleolin, RNA polymerase I, DNA topoisomerase, upstream binding factors, rRNA, rDNA, etc. in the cell nucleus are stained with silver nitrate nucleoli, which can directly display and mark the nucleolus, show the morphology and structure of the nucleolus, and determine the number, morphology, size and distribution of the nucleoli.

[0025] (3) Quantitative staining of DNA in the cell nucleus: The DNA content in the cell nucleus is closely related to the size of the cell nucleus, the chromatin texture, and the depth of the nuclear staining. The ethanol-hydrochloric acid fixative in this staining kit can depurinate the cell nuclear DNA and expose the aldehyde groups, making it easier to be stained red or purple by alkaline fuchsin, showing the chromatin texture. The depth of red or purple is proportional to the DNA content in the cell nucleus, so it can be extended to the determination of cell nuclear DNA content (DNA ploidy analysis).

[0026] (4) The key point and technical core of the present invention lies in the ability to clearly display the complete cell structure, especially to display the cell nucleus, especially to mark the nucleolus of the cell nucleus, and to clearly see the number, shape, size and distribution of the nucleoli.

[0027] (5) The staining kit and staining method of the present invention are widely used. They can be used for blood cell pathology staining and detection (including CTC, blood disease cells), as well as for exfoliated cell pathology staining and detection (including pleural effusion, ascites, cerebrospinal fluid, bronchoalveolar lavage fluid, cervical scraping, etc.), and can also be used for pathological tissue cell biopsy (including section pathological tissue cell biopsy, fine needle aspiration biopsy); in addition, the staining results of the present invention can also be extended to the determination of cell nuclear DNA content (DNA ploidy analysis). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1-4 The optical microscope photos of the staining results of Examples 1-4 are respectively shown.

[0029] Figure 5-8 The optical microscope photos of the staining results of Comparative Examples 1-4 are respectively.

[0030] Figure 9-12 The optical microscope photos of the staining results of Comparative Examples 5-8 are respectively.

[0031] Figure 13-16 The optical microscope photos of the staining results of Comparative Examples 9-12 are respectively.

[0032] Figure 17-20 The optical microscope photos of the staining results of Comparative Examples 13-16 are respectively.

[0033] Figure 21-23 The optical microscope photos of the staining results of Examples 5-7 are respectively.

[0034] Figures 24-26 The optical microscope photos of the staining results of Comparative Examples 17-19 are respectively.

[0035] Figures 27-29 The optical microscope photos of the staining results of Examples 8-10 are respectively. DETAILED DESCRIPTION

[0036] Reagents used in the specific examples:

[0037] 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.

[0038] The silver nitrate staining reagent is a silver nitrate aqueous solution with a concentration of 1-3 mol / L, and 0.2-3.0 wt% dimethyl sulfoxide is added.

[0039] The ethanol-hydrochloric acid fixative is prepared from ethanol and concentrated hydrochloric acid, wherein the concentration of the hydrochloric acid is 0.5-2 mol / L.

[0040] The gelatin formic acid reducing solution is prepared from gelatin, formic acid and water, wherein the mass concentration of gelatin is 1%-5% and the mass concentration of formic acid is 0.5-4%.

[0041] The washing solution was PBS with pH 7.2-7.4 containing Tween-20.

[0042] Schiff's reagent is prepared by mixing basic fuchsin, water, hydrochloric acid, and sodium bisulfite in a ratio of 0.5 g:100 ml:10 ml:1 g, where the concentration of hydrochloric acid is 1.5 mol / L.

[0043] HE reagent is composed of a mixture of hematoxylin staining reagent and eosin staining reagent.

[0044] Among them, the hematoxylin staining reagent is prepared from hematoxylin, ethanol, alum, water, mercuric oxide, and acetic acid in the ratio of 2.5g:25ml:2.5g:500ml:1.25g:20ml.

[0045] Among them, the eosin staining reagent is prepared from eosin, water, ethanol, and acetic acid in a ratio of 1g:75ml:25ml:1ml.

[0046] Example 1

[0047] Cytopathological staining and detection of CTC cells:

[0048] (1) Preparation of CTC cell staining detection slides;

[0049] (2) Fixing the cells with ethanol-hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treating at 370°C for 10 minutes, and rinsing with washing solution three times; wherein the hydrochloric acid concentration in the ethanol-hydrochloric acid fixative is 1.5 mol / L;

[0050] (3) mixing silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 1.5:1.0; adding the mixed reagent, staining at 370°C for 15 minutes, and rinsing with washing solution three times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L and contains 0.2 wt% dimethyl sulfoxide; the gelatin mass concentration in the gelatin formic acid reducing solution is 1%, and the mass concentration of formic acid is 2%;

[0051] (4) Add basic fuchsin staining reagent and stain at 370°C for 15 minutes;

[0052] (5) Rinse with washing solution 3 times;

[0053] (6) Allow to dry, seal with sealing tablets, and examine under an optical microscope.

[0054] Example 2

[0055] Cytopathological staining and detection of CTC cells:

[0056] (1) Preparation of CTC cell staining detection slides;

[0057] (2) Fixing the cells with ethanol-hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treating at 370°C for 10 minutes, and rinsing with washing solution three times; wherein the hydrochloric acid concentration in the ethanol-hydrochloric acid fixative is 1.5 mol / L;

[0058] (3) mixing silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 2.0:1.0; adding the mixed reagent, staining at 370°C for 15 minutes, and rinsing with washing solution three times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L and contains 0.2 wt% dimethyl sulfoxide; the gelatin mass concentration in the gelatin formic acid reducing solution is 2%, and the mass concentration of formic acid is 2%;

[0059] (4) Add basic fuchsin staining reagent and stain at 370°C for 15 minutes;

[0060] (5) Rinse with washing solution 3 times;

[0061] (6) Allow to dry, seal with sealing tablets, and examine under an optical microscope.

[0062] Example 3

[0063] Cytopathological staining and detection of CTC cells:

[0064] (1) Preparation of CTC cell staining detection slides;

[0065] (2) Fixing the cells with ethanol-hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treating at 370°C for 15 minutes, and rinsing with washing solution three times; wherein the hydrochloric acid concentration in the ethanol-hydrochloric acid fixative is 1.5 mol / L;

[0066] (3) Mixing silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 2.1:1.0; adding the mixed reagent, staining at 370°C for 15 minutes, and rinsing with washing solution three times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L and contains 0.2 wt% dimethyl sulfoxide; the gelatin mass concentration in the gelatin formic acid reducing solution is 5%, and the mass concentration of formic acid is 4%;

[0067] (4) Add basic fuchsin staining reagent and stain at 370°C for 18 minutes;

[0068] (5) Rinse with washing solution 3 times;

[0069] (6) Allow to dry, seal with sealing tablets, and examine under an optical microscope.

[0070] Example 4

[0071] Cytopathological staining and detection of CTC cells:

[0072] (1) Preparation of CTC cell staining detection slides;

[0073] (2) Fixing the cells with ethanol-hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treating at 370°C for 10-15 minutes, and rinsing with washing solution three times; wherein the hydrochloric acid concentration in the ethanol-hydrochloric acid fixative is 1.5 mol / L;

[0074] (3) mixing silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 1.5:0.9; adding the mixed reagent, staining at 370°C for 12 minutes, and rinsing with washing solution three times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L and contains 0.2 wt% dimethyl sulfoxide; the gelatin mass concentration in the gelatin formic acid reducing solution is 3%, and the mass concentration of formic acid is 3%;

[0075] (4) Add basic fuchsin staining reagent and stain at 370°C for 12 minutes;

[0076] (5) Rinse with washing solution 3 times;

[0077] (6) Allow to dry, seal with sealing tablets, and examine under an optical microscope.

[0078] The dyeing results of Examples 1-4 are shown in Figure 1-4 The cell membrane and cytoplasm are light red, the cell nucleus is dark red or purple (chromatin texture is visible), and the nucleolus is brown-black (the number, size, shape and distribution of nucleoli are revealed).

[0079] Comparative Example 1

[0080] Step (4) in Example 1 is omitted, and the other steps are the same as in Example 1.

[0081] Comparative Example 2

[0082] Step (4) in Example 2 is omitted, and the other steps are the same as in Example 2.

[0083] Comparative Example 3

[0084] Step (4) in Example 3 is omitted, and the other steps are the same as in Example 3.

[0085] Comparative Example 4

[0086] Step (4) in Example 4 is omitted, and the other steps are the same as in Example 4.

[0087] The dyeing results of Comparative Examples 1-4 are shown in Figure 5-8 .

[0088] Comparative Example 5

[0089] Step (3) in Example 1 is omitted, and the other steps are the same as in Example 1.

[0090] Comparative Example 6

[0091] Step (3) in Example 2 is omitted, and the other steps are the same as in Example 2.

[0092] Comparative Example 7

[0093] Step (3) in Example 3 is omitted, and the other steps are the same as in Example 3.

[0094] Comparative Example 8

[0095] Step (3) in Example 4 is omitted, and the other steps are the same as in Example 4.

[0096] The dyeing results of Comparative Examples 5-8 are shown in Figure 9-12 .

[0097] Comparative Example 9

[0098] The basic fuchsin staining reagent in step (4) of Example 1 was replaced with Schiff's reagent, and the other steps were the same as in Example 1.

[0099] Comparative Example 10

[0100] The basic fuchsin staining reagent in step (4) of Example 2 was replaced by Schiff's reagent, and the other steps were the same as in Example 2.

[0101] Comparative Example 11

[0102] The basic fuchsin staining reagent in step (4) of Example 3 was replaced with Schiff's reagent, and the other steps were the same as those in Example 3.

[0103] Comparative Example 12

[0104] The basic fuchsin staining reagent in step (4) of Example 4 was replaced with Schiff's reagent, and the other steps were the same as those in Example 4.

[0105] The dyeing results of Comparative Examples 9-12 are shown in Figure 13-16 .

[0106] Comparative Example 13

[0107] Step (3) in Example 1 was omitted, and the basic fuchsin staining reagent in step (4) in Example 1 was replaced with HE reagent. The other steps were the same as in Example 1.

[0108] Comparative Example 14

[0109] Step (3) in Example 2 was omitted, and the basic fuchsin staining reagent in step (4) in Example 2 was replaced by HE reagent. The other steps were the same as in Example 2.

[0110] Comparative Example 15

[0111] Step (3) in Example 3 was omitted, and the basic fuchsin staining reagent in step (4) in Example 3 was replaced by HE reagent. The other steps were the same as in Example 3.

[0112] Comparative Example 16

[0113] Step (3) in Example 4 was omitted, and the basic fuchsin staining reagent in step (4) in Example 4 was replaced by HE reagent. The other steps were the same as in Example 4.

[0114] The dyeing results of Comparative Examples 13-16 are shown in Figure 17-20 .

[0115] From the comparison of the staining results of the above embodiment and the comparative example, it can be seen that the overall staining image of the cells stained with silver nitrate-basic fuchsin of the present invention is clear; the cell membrane, cytoplasm, nucleus and nucleolus are intact and clearly defined; the cytoplasm is light red, the nucleus is red, and the nucleolus is brown-black plaque, and the number, shape and size of the nucleoli are clearly visible. However, compared with the silver nitrate reagent single staining, basic fuchsin reagent single staining, silver nitrate-Schiff reagent staining and HE reagent staining, the recognition of the various cell components is poor.

[0116] Example 5

[0117] Pathological tissue cell biopsy:

[0118] (1) Preparing pathological tissue cell detection slides;

[0119] (2) Fixing the cells with ethanol-hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treating at 370°C for 10 minutes, and rinsing with washing solution three times; wherein the hydrochloric acid concentration in the ethanol-hydrochloric acid fixative is 1.5 mol / L;

[0120] (3) mixing silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 1.5:1.0; adding the mixed reagent, staining at 370°C for 15 minutes, and rinsing with washing solution three times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L and contains 0.2 wt% dimethyl sulfoxide; the gelatin mass concentration in the gelatin formic acid reducing solution is 1%, and the mass concentration of formic acid is 2%;

[0121] (4) Add basic fuchsin staining reagent and stain at 370°C for 15 minutes;

[0122] (5) Rinse with washing solution 3 times;

[0123] (6) Allow to dry, seal with sealing tablets, and examine under an optical microscope.

[0124] Example 6

[0125] Pathological tissue cell biopsy:

[0126] (1) Preparing pathological tissue cell detection slides;

[0127] (2) Fixing the cells with ethanol-hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treating at 370°C for 10 minutes, and rinsing with washing solution three times; wherein the hydrochloric acid concentration in the ethanol-hydrochloric acid fixative is 1.5 mol / L;

[0128] (3) mixing silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 2.0:1.0; adding the mixed reagent, staining at 370°C for 15 minutes, and rinsing with washing solution three times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L and contains 0.2 wt% dimethyl sulfoxide; the gelatin mass concentration in the gelatin formic acid reducing solution is 2%, and the mass concentration of formic acid is 2%;

[0129] (4) Add basic fuchsin staining reagent and stain at 370°C for 15 minutes;

[0130] (5) Rinse with washing solution 3 times;

[0131] (6) Allow to dry, seal with sealing tablets, and examine under an optical microscope.

[0132] Example 7

[0133] Pathological tissue cell biopsy:

[0134] (1) Preparing pathological tissue cell detection slides;

[0135] (2) Fixing the cells with ethanol-hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treating at 370°C for 15 minutes, and rinsing with washing solution three times; wherein the hydrochloric acid concentration in the ethanol-hydrochloric acid fixative is 1.5 mol / L;

[0136] (3) Mixing silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 2.1:1.0; adding the mixed reagent, staining at 370°C for 15 minutes, and rinsing with washing solution three times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L and contains 0.2 wt% dimethyl sulfoxide; the gelatin mass concentration in the gelatin formic acid reducing solution is 5%, and the mass concentration of formic acid is 4%;

[0137] (4) Add basic fuchsin staining reagent and stain at 370°C for 18 minutes;

[0138] (5) Rinse with washing solution 3 times;

[0139] (6) Allow to dry, seal with sealing tablets, and examine under an optical microscope.

[0140] Comparative Example 17

[0141] Step (3) in Example 5 was omitted, and the basic fuchsin staining reagent in step (4) in Example 5 was replaced by HE reagent. The other steps were the same as in Example 5.

[0142] Comparative Example 18

[0143] Step (3) in Example 6 was omitted, and the basic fuchsin staining reagent in step (4) in Example 6 was replaced by HE reagent. The other steps were the same as in Example 6.

[0144] Comparative Example 19

[0145] Step (3) in Example 7 was omitted, and the basic fuchsin staining reagent in step (4) in Example 7 was replaced by HE reagent. The other steps were the same as in Example 7.

[0146] The dyeing results of Examples 5-7 are shown in Figure 21-23 The dyeing results of comparative examples 17-19 are shown in Figures 24-26 From the comparison of the staining results of the above six paraffin sections of pathological tissues, it can be seen that HE staining can only display general cell nucleus images, but the images of the cell nucleoli are quite blurred and have low resolution; however, the staining method of the present invention can not only clearly display the complete cell nucleus, but also clearly display the number, morphology, size and distribution of the cell nucleoli.

[0147] Example 8

[0148] Cytopathological staining and detection of CTC cells:

[0149] (1) Preparation of CTC cell staining detection slides;

[0150] (2) Fixing the cells with ethanol-hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treating at 370°C for 10 minutes, and rinsing with washing solution three times; wherein the hydrochloric acid concentration in the ethanol-hydrochloric acid fixative is 0.5 mol / L;

[0151] (3) mixing silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 1.5:1.0; adding the mixed reagent, staining at 370°C for 15 minutes, and rinsing with washing solution three times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L and contains 0.2 wt% dimethyl sulfoxide; the gelatin mass concentration in the gelatin formic acid reducing solution is 1%, and the mass concentration of formic acid is 2%;

[0152] (4) Add basic fuchsin staining reagent and stain at 370°C for 15 minutes;

[0153] (5) Rinse with washing solution 3 times;

[0154] (6) Allow to dry, seal with sealing tablets, and examine under an optical microscope.

[0155] Example 9

[0156] The hydrochloric acid concentration in the ethanol hydrochloric acid fixative in step (2) of Example 8 was replaced with 1.5 mol / L, and the other steps were the same as in Example 8.

[0157] Example 10

[0158] The hydrochloric acid concentration in the ethanol hydrochloric acid fixative in step (2) of Example 8 was replaced with 2.0 mol / L, and the other steps were the same as in Example 8.

[0159] The dyeing results of Examples 8-10 are shown in Figures 27-29 By comparison, it can be found that the final staining effect of the ethanol hydrochloric acid fixative with a hydrochloric acid concentration of 1.5 mol / L is the best.

[0160] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cytopathology staining kit, characterized in that: It includes basic fuchsin staining reagent, silver nitrate staining reagent, ethanol hydrochloric acid fixative, gelatin formic acid reducing solution and washing solution; 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; The silver nitrate staining reagent is a silver nitrate aqueous solution with a concentration of 1-3 mol / L, and 0.2-3.0 wt% dimethyl sulfoxide is added; The washing solution is PBS with a pH of 7.2-7.4 and containing Tween-20.

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

3. The cytopathology staining kit according to claim 1, characterized in that The gelatin formic acid reduction solution is prepared from gelatin, formic acid and water, wherein the mass concentration of gelatin is 1%-5%, and the mass concentration of formic acid is 0.5-4%.

4. A cytopathological staining method, characterized in that: The cytopathology staining kit according to any one of claims 1 to 3 is used, comprising the following steps: (1) Preparation of human tissue cell specimens; (2) Fix the cells with ethanol hydrochloric acid fixative to expose aldehyde groups in the nuclear DNA, treat at 370°C for 10-15 minutes, and rinse three times with washing solution; (3) Mix the silver nitrate staining reagent and gelatin formic acid reducing solution in a volume ratio of 1.5-2.1:0.9-1.2; add the mixed reagent, stain at 370°C for 12-18 minutes, and rinse with washing solution 3 times; (4) Add basic fuchsin staining reagent and stain at 370°C for 12-18 minutes; (5) Rinse with detergent 3 times; (6) Allow to dry, seal the slides with sealing tablets, and examine under an optical microscope.

Images