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-hydrochloric acid fixation solution, a gelatin-formic acid reducing solution, and a washing solution. The kit can reveal the complete cellular structure and morphology thereof, and can particularly delineate the morphology of a nucleolus of a cell nucleus.

Description

Cell pathology staining kit and staining method

[0001] The present application claims priority to the Chinese patent application No. 202410762100.1, filed on June 13, 2024, and entitled "A cell pathology staining kit and staining method", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of pathological examination and analysis, and specifically relates to a cell pathology staining kit and staining method. BACKGROUND

[0003] Human cells are composed of three parts: cell membrane, cytoplasm (cytoplasm) and nucleus, but cell pathology should pay special attention to the structure and morphology of the nucleus and nucleolus in the pathological cells. The nucleolus in the nucleus is a vital organelle in the nucleus and is the hub of all cell activities. The change of the nucleolus in the nucleus is the basis and core evidence for identifying the pathological structure and morphology of malignant cells. The nucleolus contains nucleolus markers such as nucleolar fibrillarin, nucleolar phosphoprotein, nucleolin, RNA polymerase I, DNA topoisomerase, upstream binding factor and rRNA, rDNA, etc. There are obvious differences in the morphological structure of the nucleus and nucleolus between normal cells and malignant cells.

[0004] At present, the staining methods or techniques for detecting malignant cells or pathological tissues mainly include four kinds: histopathology examination, bone marrow cytology examination, exfoliated cell examination and peripheral blood circulating tumor cell (CTC) liquid biopsy. Among them,

[0005] Histopathology examination mainly uses HE and histochemical staining to detect and identify malignant cells, and can only see the basic structure of cells, such as cell membrane, cytoplasm and cell morphological structure of nucleus. Its main technical defect is that it cannot clearly show the morphology of the nucleolus, cannot clearly mark the nucleolus, and cannot distinguish the nucleolus from the nucleoplasm.

[0006] The chemical staining method used in exfoliated cell examination has basically the same results as the histopathology examination method, which can show the basic structure of cells, can show the cell nucleus, but cannot clearly show the morphological structure of the nucleolus, cannot mark the nucleolus, and cannot distinguish the nucleolus from the nucleoplasm.

[0007] Bone marrow cytology examination uses chemical staining, which can show the overall structure of cells, show the nucleus and nucleolus, but bone marrow cytology uses general chemical staining, which has low sensitivity to nucleolus display, and cannot distinguish the nucleolus from the nucleoplasm, which brings some uncertainty to the cytological diagnosis of blood diseases.

[0008] The chemical staining of the current CTC cytological detection has poor repeatability and low sensitivity, and the nucleolus morphology cannot be clearly shown; the mainstream immunofluorescence staining method focuses on showing the cell surface antigen, and cannot show the morphology and structure of the nucleolus of the cell nucleus, and the cell nucleus is stained by the antigen or DAPI to determine whether it is a CTC cell, and false positives or false negatives may occur due to changes or illegal editing of the antigen. CTC cannot be checked and identified from cell pathology characteristics. SUMMARY

[0009] In order to overcome some deficiencies in the prior art, the application provides a cell pathology staining kit and a staining method, which can clearly show the morphology of the cell membrane, cytoplasm and cell nucleus, especially the nucleolus of the cell nucleus.

[0010] The first aspect of the application provides a cell pathology staining kit, which comprises an alkaline magenta staining reagent, a silver nitrate staining reagent, an ethanol hydrochloric acid fixing solution, a gelatin formic acid reducing solution and a washing solution.

[0011] In an embodiment, the alkaline magenta staining reagent is prepared by alkaline magenta, acetic acid and water.

[0012] In an embodiment, the alkaline magenta staining reagent is prepared by alkaline magenta, acetic acid and water in a ratio of 0.1 (g):1 (ml):100 (ml).

[0013] In an embodiment, the silver nitrate staining reagent is a silver nitrate aqueous solution; the concentration is 1-3 mol / L.

[0014] In an embodiment, the ethanol hydrochloric acid fixing solution is prepared by ethanol and hydrochloric acid; the concentration of hydrochloric acid in the ethanol hydrochloric acid fixing solution is 0.5-2 mol / L.

[0015] In an embodiment, the ethanol hydrochloric acid fixing solution is prepared by ethanol and concentrated hydrochloric acid; the concentration of hydrochloric acid in the ethanol hydrochloric acid fixing solution is 0.5-2 mol / L.

[0016] In an embodiment, the gelatin formic acid reducing solution is prepared by gelatin, formic acid and water; the mass ratio of gelatin in the gelatin formic acid reducing solution is 1%-5%, and the mass ratio of formic acid is 0.5-4%.

[0017] In an embodiment, the volume ratio of the silver nitrate staining reagent to the gelatin formic acid reducing solution is 1.5-2.1:0.9-1.2.

[0018] In an embodiment, the washing solution is a PBS containing Tween-20 and having a pH of 7.2-7.4.

[0019] The second aspect of the present application provides a method for cytopathological staining, using the cytopathological staining kit of any one of the preceding embodiments, comprising the following steps:

[0020] (1) preparing a human tissue cell specimen;

[0021] (2) fixing the cells in the specimen with an ethanol hydrochloric acid fixing solution and exposing the aldehyde groups of the DNA in the cell nucleus, and then rinsing with a washing solution;

[0022] (3) mixing the silver nitrate staining reagent and the gelatin formic acid reducing solution to obtain a mixed reagent, adding the mixed reagent, and then rinsing with a washing solution;

[0023] (4) adding the basic fuchsin staining reagent, and then rinsing with a washing solution;

[0024] (5) air-drying, adding a mounting agent for mounting, and then observing under an optical microscope.

[0025] More specifically, the staining method comprises:

[0026] (1) preparing or preparing a human tissue cell specimen;

[0027] (2) fixing the cells in the specimen with an ethanol hydrochloric acid fixing solution and exposing the aldehyde groups of the DNA in the cell nucleus, and then rinsing with a washing solution;

[0028] (3) mixing the silver nitrate staining reagent and the gelatin formic acid reducing solution according to a volume ratio of 1.5-2.1:0.9-1.2, adding the mixed reagent, and then rinsing with a washing solution;

[0029] (4) adding the basic fuchsin staining reagent, and then rinsing with a washing solution;

[0030] (5) air-drying, adding a mounting agent for mounting, and then observing under an optical microscope.

[0031] The third aspect of the present application provides an application of the cytopathological staining kit of any one of the preceding embodiments, which can stain the cell membrane, the cytoplasm and the cell nucleus of a cell to show the cell membrane, the cytoplasm, and the morphology of the cell nucleus. In an embodiment, the morphology of the nucleolus of the cell nucleus can be further shown.

[0032] As known to those skilled in the art, the morphology mainly refers to the shape, size, structure and dynamic changes of the nucleus, and the nucleolus mainly refers to the shape, size, number, structural partition and dynamic changes of the nucleolus. In the present application, silver nitrate and basic fuchsin staining not only shows the color difference between the cytoplasm and the nucleus, but also clearly and completely shows the morphology of the nucleolus of the nucleus.

[0033] The fourth aspect of the present application provides an application of the cytopathology staining kit of any one of the preceding embodiments, for staining and identifying normal cells and pathological cells.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] (1) The cytopathology staining kit and staining method provided by at least one embodiment of the present application can show complete cell structures: the combination of silver nitrate staining reagent and basic fuchsin staining reagent makes the complete cell structures of cell membrane, cytoplasm, nucleus and nucleolus clearly visible after staining; in particular, the morphology of the nucleolus of the nucleus is shown, including the shape, number, size and distribution of the nucleolus.

[0036] (2) The cytopathology staining kit and staining method provided by at least one embodiment of the present application can show the structure and morphology of the nucleolus in the nucleus: the nucleolus markers (nucleolus internal substances) in the nucleus, such as nucleolar fibrillarin, nucleolar phosphoprotein, nucleolin, RNA polymerase I, DNA topoisomerase, upstream binding factor and rRNA, rDNA, etc. are stained by silver nitrate staining reagent, which can directly show and calibrate the nucleolus and show and determine the morphology of the nucleolus.

[0037] (3) The cytopathology staining kit and staining method provided by at least one embodiment of the present application can quantitatively stain the DNA in the nucleus: the DNA content in the nucleus is closely related to the size of the nucleus, the chromatin texture and the staining depth of the nucleus. The ethanol hydrochloric acid fixing solution in the staining kit can make the DNA deprotonation in the nucleus expose the aldehyde group more easily to be stained red or purple red by basic fuchsin, showing the chromatin texture, and the depth of red or purple red is proportional to the DNA content in the nucleus, so it can be extended for DNA content determination (DNA ploidy analysis).

[0038] (4) The cytopathology staining kit and staining method provided by at least one embodiment of the present application can show clear and complete cell structures, in particular, the nucleolus of the nucleus can be calibrated, and the morphology of the nucleolus is clearly visible.

[0039] (5) The cell pathology staining kit and staining method provided by at least one embodiment of the present application have wide applications, and can be applied to blood cell pathology staining and detection (including CTC, blood disease cells), exfoliated cell pathology staining and detection (including chest fluid, abdominal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, cervical scraping, etc.), and pathological tissue biopsy (including section pathological tissue biopsy, fine needle biopsy), and the staining result of the present application can also be extended to cell nucleus DNA content determination (DNA ploidy analysis). BRIEF DESCRIPTION OF DRAWINGS

[0040] Fig. 1-4 are optical microscope photos of the staining results of Examples 1-4, respectively;

[0041] Fig. 5-8 are optical microscope photos of the staining results of Comparative Examples 1-4, respectively;

[0042] Fig. 9-12 are optical microscope photos of the staining results of Comparative Examples 5-8, respectively;

[0043] Fig. 13-16 are optical microscope photos of the staining results of Comparative Examples 9-12, respectively;

[0044] Fig. 17-20 are optical microscope photos of the staining results of Comparative Examples 13-16, respectively;

[0045] Fig. 21-23 are optical microscope photos of the staining results of Examples 5-7, respectively;

[0046] Fig. 24-26 are optical microscope photos of the staining results of Comparative Examples 17-19, respectively;

[0047] Fig. 27-29 are optical microscope photos of the staining results of Examples 8-10, respectively. DETAILED DESCRIPTION

[0048] The present application will be described in detail below in combination with examples, and it is worth understanding that these examples are only preferred embodiments of the present application, and cannot be understood as limiting the protection scope of the present application.

[0049] The reagents used in the embodiments of the present application have the following specific compositions:

[0050] The basic fuchsin staining reagent is prepared by mixing basic fuchsin (chemical formula: C 19 H 18 ClN3), acetic acid and water according to the ratio of 0.1 g: 1 ml: 100 ml.

[0051] The silver nitrate staining reagent is an aqueous silver nitrate solution with a concentration of 1-3 mol / L (for example, 1.5 mol / L, 2 mol / L, 2.5 mol / L, etc.), and 0.2-3.0 wt% dimethyl sulfoxide is added, i.e., the mass fraction of dimethyl sulfoxide in the silver nitrate staining reagent is 0.2-3.0%.

[0052] The ethanol hydrochloric acid fixing solution is prepared from ethanol and concentrated hydrochloric acid, wherein the concentration of hydrochloric acid in the ethanol hydrochloric acid fixing solution is 0.5-2 mol / L, for example, 1 mol / L, 1.5 mol / L, etc.

[0053] The gelatin formic acid reducing solution is prepared from gelatin, formic acid and water; wherein the mass concentration of gelatin is 1%-5%, i.e., the mass fraction of gelatin in the gelatin formic acid reducing solution is 1%-5%, for example, 2%, 3%, 4%, etc.; the mass concentration of formic acid is 0.5-4%, i.e., the mass fraction of formic acid in the gelatin formic acid reducing solution is 0.5-4%, for example, 1%, 2%, 3%, etc.

[0054] The washing solution is a PBS (Phosphate Buffered Saline) with Tween-20 and a pH of 7.2-7.4.

[0055] The Schiff reagent is prepared from basic fuchsin, water, hydrochloric acid and sodium bisulfite according to a ratio of 0.5 g:100 ml:10 ml:1 g, wherein the concentration of hydrochloric acid is 1.5 mol / L.

[0056] The HE reagent is composed of a hematoxylin staining reagent and an eosin staining reagent. The hematoxylin staining reagent is prepared from hematoxylin, ethanol, alum, water, mercuric oxide and acetic acid according to 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 according to a ratio of 1 g:75 ml:25 ml:1 ml.

[0057] Example 1: CTC cell cytopathological staining and detection:

[0058] (1) Preparation of CTC cell staining and detection slices;

[0059] (2) Fixing the cells with the ethanol hydrochloric acid fixing solution to expose the aldehyde groups of the cell nucleus DNA, treating at 37°C for 10 minutes, and rinsing with the washing solution for 3 times; wherein the concentration of hydrochloric acid in the ethanol hydrochloric acid fixing solution is 1.5 mol / L;

[0060] (3) Mix the silver nitrate staining reagent and the gelatin formic acid reducing solution according to a volume ratio of 1.5:1.0; add the mixed reagent, and dye for 15 minutes at 37°C, and then rinse with the washing solution for 3 times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L, and the silver nitrate staining reagent contains 0.2 wt% dimethyl sulfoxide; the gelatin mass ratio in the gelatin formic acid reducing solution is 1%, and the formic acid mass ratio is 2%;

[0061] (4) add the basic fuchsin staining reagent, dye for 15 minutes at 37°C, and then rinse with the washing solution for 3 times;

[0062] (5) air dry, add the mounting agent for mounting, and then perform optical microscope examination.

[0063] Example 2: CTC cell cytopathological staining and detection:

[0064] (1) prepare a CTC cell staining and detection slice;

[0065] (2) fix the cells with the ethanol hydrochloric acid fixing solution and expose the aldehyde groups of the cell nucleus DNA, and then treat at 37°C for 10 minutes, and then rinse with the washing solution for 3 times; wherein the hydrochloric acid concentration in the ethanol hydrochloric acid fixing solution is 1.5 mol / L;

[0066] (3) mix the silver nitrate staining reagent and the gelatin formic acid reducing solution according to a volume ratio of 2.0:1.0; add the mixed reagent, and dye for 15 minutes at 37°C, and then rinse with the washing solution for 3 times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L, and the silver nitrate staining reagent contains 0.2 wt% dimethyl sulfoxide; the gelatin mass ratio in the gelatin formic acid reducing solution is 2%, and the formic acid mass ratio is 2%;

[0067] (4) add the basic fuchsin staining reagent, dye for 15 minutes at 37°C, and then rinse with the washing solution for 3 times;

[0068] (5) air dry, add the mounting agent for mounting, and then perform optical microscope examination.

[0069] Example 3: CTC cell cytopathological staining and detection:

[0070] (1) prepare a CTC cell staining and detection slice;

[0071] (2) fix the cells with the ethanol hydrochloric acid fixing solution and expose the aldehyde groups of the cell nucleus DNA, and then treat at 37°C for 15 minutes, and then rinse with the washing solution for 3 times; wherein the hydrochloric acid concentration in the ethanol hydrochloric acid fixing solution is 1.5 mol / L;

[0072] (3) mixing silver nitrate staining reagent and gelatin formic acid reducing solution according to the volume ratio of 2.1:1.0; adding the mixed reagent, staining at 37℃ for 15 minutes, and rinsing with washing solution for 3 times; wherein, the concentration of silver nitrate in silver nitrate staining reagent is 1 mol / L, and 0.2wt% dimethyl sulfoxide is contained; the mass ratio of gelatin in gelatin formic acid reducing solution is 5%, and the mass ratio of formic acid is 4%;

[0073] (4) adding basic fuchsin staining reagent, staining at 37℃ for 18 minutes, and rinsing with washing solution for 3 times;

[0074] (5) air-drying, adding mounting medium for mounting, and observing under optical microscope.

[0075] Example 4: CTC cell cytopathological staining and detection:

[0076] (1) preparing CTC cell staining detection slide;

[0077] (2) fixing cells with ethanol hydrochloric acid fixing solution and exposing aldehyde group of cell nucleus DNA, treating at 37℃ for 10-15 minutes, and rinsing with washing solution for 3 times; wherein, the concentration of hydrochloric acid in ethanol hydrochloric acid fixing solution is 1.5 mol / L;

[0078] (3) mixing silver nitrate staining reagent and gelatin formic acid reducing solution according to the volume ratio of 1.5:0.9; adding the mixed reagent, staining at 37℃ for 12 minutes, and rinsing with washing solution for 3 times; wherein, the concentration of silver nitrate in silver nitrate staining reagent is 1 mol / L, and 0.2wt% dimethyl sulfoxide is contained; the mass ratio of gelatin in gelatin formic acid reducing solution is 3%, and the mass ratio of formic acid is 3%;

[0079] (4) adding basic fuchsin staining reagent, staining at 37℃ for 12 minutes, and rinsing with washing solution for 3 times;

[0080] (5) air-drying, adding mounting medium for mounting, and observing under optical microscope.

[0081] The optical microscope photos of the staining results of Examples 1-4 are shown in Figures 1-4 respectively; wherein, the cell membrane and cytoplasm are light red; the cell nucleus is dark red or purple red, and chromatin texture can be seen; the nucleolus of cell nucleus is brown black, and the number, size, morphology and distribution of nucleolus are shown.

[0082] Comparative Example 1: omitting step (4) in Example 1, and other steps are the same as Example 1.

[0083] Comparative Example 2: omitting step (4) in Example 2, and other steps are the same as Example 2.

[0084] Comparative Example 3: omitting step (4) in Example 3, and other steps are the same as Example 3.

[0085] Comparative Example 4: Step (4) in Example 4 was omitted, and the other steps were the same as in Example 4.

[0086] The staining results of Comparative Examples 1-4 are shown in Figures 5-8, respectively.

[0087] Comparative Example 5: Step (3) in Example 1 was omitted, and the other steps were the same as in Example 1.

[0088] Comparative Example 6: Step (3) in Example 2 was omitted, and the other steps were the same as in Example 2.

[0089] Comparative Example 7: Step (3) in Example 3 was omitted, and the other steps were the same as in Example 3.

[0090] Comparative Example 8: Step (3) in Example 4 was omitted, and the other steps were the same as in Example 4.

[0091] The optical microscope photos of the staining results of Comparative Examples 5-8 are shown in Figures 9-12, respectively.

[0092] Comparative Example 9: The basic fuchsin staining reagent in Step (4) in Example 1 was replaced with Schiff reagent, and the other steps were the same as in Example 1.

[0093] Comparative Example 10: The basic fuchsin staining reagent in Step (4) in Example 2 was replaced with Schiff reagent, and the other steps were the same as in Example 2.

[0094] Comparative Example 11: The basic fuchsin staining reagent in Step (4) in Example 3 was replaced with Schiff reagent, and the other steps were the same as in Example 3.

[0095] Comparative Example 12: The basic fuchsin staining reagent in Step (4) in Example 4 was replaced with Schiff reagent, and the other steps were the same as in Example 4.

[0096] The optical microscope photos of the staining results of Comparative Examples 9-12 are shown in Figures 13-16, respectively.

[0097] Comparative Example 13: Step (3) in Example 1 was omitted, and the basic fuchsin staining reagent in Step (4) in Example 1 was replaced with HE reagent, and the other steps were the same as in Example 1.

[0098] Comparative Example 14: Step (3) in Example 2 was omitted, and the basic fuchsin staining reagent in Step (4) in Example 2 was replaced with HE reagent, and the other steps were the same as in Example 2.

[0099] Comparative Example 15: Step (3) in Example 3 was omitted, and the basic fuchsin staining reagent in Step (4) in Example 3 was replaced with HE reagent, and the other steps were the same as in Example 3.

[0100] Comparative Example 16: Step (3) in Example 4 was omitted, and the basic fuchsin staining reagent in step (4) in Example 4 was replaced by HE reagent, and the other steps were the same as in Example 4.

[0101] The optical microscope photos of the staining results of Comparative Examples 13-16 are shown in Figures 17-20, respectively.

[0102] By comparing the photos of the staining results of Examples 1-4 (Figures 1-4) and the photos of the staining results of Comparative Examples 1-16 (Figures 5-20), it can be seen that the overall staining image of the cells was clear, the cell membrane, cytoplasm, nucleus and nucleolus were complete and had distinct boundaries, the cytoplasm was light red, the nucleus was red, and the nucleolus was brown-black patches, and the morphology of the nucleolus, including the number and size, was clearly visible. In comparison, the identification of the various components of the cells was poorer in the case of single staining with silver nitrate reagent, single staining with basic fuchsin reagent, staining with silver nitrate-Shiff reagent, and staining with HE reagent, and the silver nitrate-basic fuchsin reagent staining of the present application.

[0103] Example 5: Pathological tissue cell biopsy

[0104] (1) Preparation of pathological tissue cell detection sheet;

[0105] (2) Fix the cells with ethanol hydrochloric acid fixing solution and expose the aldehyde group of the DNA of the cell nucleus, treat at 37°C for 10 minutes, and rinse with washing liquid for 3 times; wherein the concentration of hydrochloric acid in the ethanol hydrochloric acid fixing solution is 1.5 mol / L;

[0106] (3) Mix the silver nitrate staining reagent and the gelatin formic acid reducing solution in a volume ratio of 1.5:1.0; add the mixed reagent, and stain at 37°C for 15 minutes, and rinse with washing liquid for 3 times; wherein the concentration of silver nitrate in the silver nitrate staining reagent is 1 mol / L, and it contains 0.2 wt% dimethyl sulfoxide; the mass ratio of gelatin in the gelatin formic acid reducing solution is 1%, and the mass ratio of formic acid is 2%;

[0107] (4) Add the basic fuchsin staining reagent, and stain at 37°C for 15 minutes, and rinse with washing liquid for 3 times;

[0108] (5) Air dry, add mounting medium for mounting, and examine under an optical microscope.

[0109] Example 6: Pathological tissue cell biopsy

[0110] (1) Preparation of pathological tissue cell detection sheet;

[0111] (2) Fix the cells with ethanol hydrochloric acid fixing solution and expose the aldehyde group of the DNA of the cell nucleus, treat at 37°C for 10 minutes, and rinse with washing liquid for 3 times; wherein the concentration of hydrochloric acid in the ethanol hydrochloric acid fixing solution is 1.5 mol / L;

[0112] (3) Mix the silver nitrate staining reagent and the gelatin formic acid reducing solution according to a volume ratio of 2.0:1.0; add the mixed reagent, and dye for 15 minutes at 37°C, and then rinse with the washing solution for 3 times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L, and the silver nitrate staining reagent contains 0.2wt% dimethyl sulfoxide; the gelatin mass ratio in the gelatin formic acid reducing solution is 2%, and the formic acid mass ratio is 2%;

[0113] (4) add the basic fuchsin staining reagent, dye for 15 minutes at 37°C, and then rinse with the washing solution for 3 times;

[0114] (5) air dry, add the mounting agent to mount, and then observe under the optical microscope.

[0115] Example 7: pathological tissue cell biopsy

[0116] (1) prepare the pathological tissue cell detection slice;

[0117] (2) fix the cells with the ethanol hydrochloric acid fixing solution to expose the aldehyde groups of the cell nucleus DNA, and then treat at 37°C for 15 minutes, and then rinse with the washing solution for 3 times; wherein the hydrochloric acid concentration in the ethanol hydrochloric acid fixing solution is 1.5 mol / L;

[0118] (3) mix the silver nitrate staining reagent and the gelatin formic acid reducing solution according to a volume ratio of 2.1:1.0; add the mixed reagent, and dye for 15 minutes at 37°C, and then rinse with the washing solution for 3 times; wherein the silver nitrate concentration in the silver nitrate staining reagent is 1 mol / L, and the silver nitrate staining reagent contains 0.2wt% dimethyl sulfoxide; the gelatin mass ratio in the gelatin formic acid reducing solution is 5%, and the formic acid mass ratio is 4%;

[0119] (4) add the basic fuchsin staining reagent, dye for 18 minutes at 37°C, and then rinse with the washing solution for 3 times;

[0120] (5) air dry, add the mounting agent to mount, and then observe under the optical microscope.

[0121] Comparative Example 17: omit step (3) in Example 5, replace the basic fuchsin staining reagent in step (4) in Example 5 with the HE reagent, and the other steps are the same as those in Example 5.

[0122] Comparative Example 18: omit step (3) in Example 6, replace the basic fuchsin staining reagent in step (4) in Example 6 with the HE reagent, and the other steps are the same as those in Example 6.

[0123] Comparative Example 19: omit step (3) in Example 7, replace the basic fuchsin staining reagent in step (4) in Example 7 with the HE reagent, and the other steps are the same as those in Example 7.

[0124] The optical microscope photos of the staining results of Examples 5-7 are shown in Figures 21-23 respectively, and the optical microscope photos of the staining results of Comparative Examples 17-19 are shown in Figures 24-26 respectively. By comparing the above six photos of the staining results of the pathological paraffin sections, it can be seen that: the HE staining can only show the general image of the cell nucleus, but the image of the nucleolus of the cell nucleus is quite blurred and has low resolution; but the staining method of the present application can clearly show the complete cell nucleus and the morphology of the cell nucleolus, including the number, size and distribution characteristics.

[0125] Example 8: CTC cell cytopathological staining and detection:

[0126] (1) Preparation of CTC cell staining and detection section;

[0127] (2) Fix the cells with ethanol hydrochloric acid fixing solution and expose the aldehyde group of the cell nucleus DNA, treat at 37°C for 10 minutes, and rinse with washing solution for 3 times; wherein the concentration of hydrochloric acid in the ethanol hydrochloric acid fixing solution is 0.5 mol / L;

[0128] (3) Mix the silver nitrate staining reagent and the gelatin formic acid reducing solution according to the volume ratio of 1.5:1.0; add the mixed reagent, stain at 37°C for 15 minutes, and rinse with washing solution for 3 times; wherein the concentration of silver nitrate in the silver nitrate staining reagent is 1 mol / L, and 0.2wt% dimethyl sulfoxide is contained; the mass ratio of gelatin in the gelatin formic acid reducing solution is 1%, and the mass ratio of formic acid is 2%;

[0129] (4) Add the basic fuchsin staining reagent, stain at 37°C for 15 minutes, and rinse with washing solution for 3 times;

[0130] (5) Air dry, add mounting medium for mounting, and examine under an optical microscope.

[0131] Example 9: Replace the concentration of hydrochloric acid in the ethanol hydrochloric acid fixing solution in step (2) of Example 8 with 1.5 mol / L, and the other steps are the same as those in Example 8.

[0132] Example 10: Replace the concentration of hydrochloric acid in the ethanol hydrochloric acid fixing solution in step (2) of Example 8 with 2.0 mol / L, and the other steps are the same as those in Example 8.

[0133] The optical microscope photos of the staining results of Examples 8-10 are shown in Figures 27-29 respectively, and by comparison, it can be found that the final staining effect is best when the ethanol hydrochloric acid fixing solution with a hydrochloric acid concentration of 1.5 mol / L is used.

[0134] The embodiments described above are merely preferred embodiments of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A kit for cytopathological staining, wherein, The basic fuchsin staining reagent, the silver nitrate staining reagent, the ethanol hydrochloric acid fixing solution, the gelatin formic acid reducing solution and the washing solution are included.

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

3. The cytopathological staining kit according to claim 1, wherein, The basic fuchsin staining reagent is prepared by using 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 a silver nitrate aqueous solution with a concentration of 1-3 mol / L.

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

6. The cytopathological staining kit according to claim 1, wherein, The gelatin formic acid reducing solution is prepared by using gelatin, formic acid and water, and the mass ratio of gelatin in the gelatin formic acid reducing solution is 1%-5%, and the mass ratio of formic acid is 0.5-4%.

7. The cytopathological staining kit according to claim 1, wherein, The 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 a PBS with Tween-20 and a pH of 7.2-7.

4.

9. A method of cytopathological staining, wherein, The cytopathology staining kit of any one of claims 1-8 comprises the following steps: (1) preparing a human tissue cell specimen; (2) fixing the cells in the specimen with the ethanol hydrochloric acid fixing solution to expose the aldehyde groups of the cell nucleus DNA, and then washing with the washing solution; (3) mixing the silver nitrate staining reagent and the gelatin formic acid reducing solution to obtain a mixed reagent, and then adding the mixed reagent and washing with the washing solution after treatment; (4) adding the basic fuchsin staining reagent, and then washing with the washing solution after treatment; (5) drying and sealing with a sealing agent, and then observing under an optical microscope.

10. The cytopathology staining kit of any one of claims 1-8 is used to stain the cell membrane, cytoplasm and cell nucleus of cells, and can show the morphology of the nucleolus of the cell nucleus.

11. The cytopathology staining kit of any one of claims 1-8 is used to identify normal cells and pathological cells.

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