CN114839360A - Human exfoliative cell immunofluorescence staining kit and method - Google Patents

Abstract

The invention discloses a kit and a method for fluorescent staining of human exfoliated cells, which are characterized in that a fluorescent dye SYTO RNA Select with special affinity to cell nuclei, particularly cell nucleolus RNA is used for staining the cell nucleolus and the cell nucleus to be green, monoclonal antibodies of epithelial cell antigens and the like (EpCAM, CK, vimentin and the like) are combined to mark red fluorescent dye, and cytoplasm and the cell surface are stained to be red. The staining method and the staining kit can clearly and completely display the cell nucleus, particularly the nucleolus which can mark the cell nucleus, and clearly show the quantity, the shape, the volume size and the distribution of the nucleolus. The key characteristic of human cytopathology is analyzed, and the antigen of cytoplasm and cell membrane is displayed by immunofluorescence staining method, so that the full-form observation and analysis of human exfoliative cells can be carried out.

Description

Human exfoliative cell immunofluorescence staining kit and method

Technical Field

The invention relates to the field of medical inspection and malignant cell analysis, in particular to a fluorescent staining reagent and a staining method for human exfoliated cells including Circulating Tumor Cells (CTC) with solid tumor cells exfoliated and entering blood circulation, exfoliated pleural effusion, exfoliated cerebrospinal fluid, exfoliated vaginal cervix cells, exfoliated urethra, exfoliated gastrointestinal tract cells and blood disease cells.

Background

Human cells are divided into four parts, namely cell membranes, cytoplasm (cytoplasm), cell nuclei and nucleoli, wherein the nucleoli is the key of cell growth, development and differentiation and proliferation, malignant tumor lesions are just the cell differentiation and development problems, and the nucleoli which is the first pathological change in the processes of the attack of malignant tumors and the disease progression is the cell nucleoli, so that the pathological morphological changes of the cell nuclei and the nucleoli are emphasized in the pathological examination of the malignant tumor cells, and the changes of the cell nuclei, particularly the nucleoli, are the basis for identifying the pathological morphology of the malignant tumor cells.

Under an electron microscope, the nucleoli in the nucleus is structurally classified as: (1) fiber center: the fiber center is the innermost structure of the kernel and accounts for 5 percent of the total volume of the kernel. Is the distribution region and locus of nucleolar fibrin, nucleolar protein, nucleolar phosphoprotein, RNA polymerase I, etc. of nucleolar markers. The number, area size, morphology and the like of the fiber centers are closely related to the metabolic activity, growth speed and the like of cells, and the fiber centers are actually equivalent to the nucleolar centers of cell nuclei, so that the number, area size, morphology and distribution of the fiber centers are closely related to the malignant pathological changes of the cells. (2) The fiber component is composed of fiber filaments of 4-10nm, and accounts for about 15% of the total volume of the kernel. It is first formed to be closely attached to and surround the center of the fiber as the nucleolus occurs. (3) The particle components are as follows: around the fiber component, the fiber component accounts for about 75 percent of the compact type core.

The traditional histopathological examination, the exfoliative cytological examination and the blood cell examination mainly use chemical staining for many years, can see the basic structures of malignant tumor cells, such as the morphological structures of cell membranes, cell paddles and cell nuclei, and show the partial cytopathological characteristics of nuclear enlargement, nuclear malformation and nuclear plasma ratio disorder of the malignant cells; the main technical defects of the traditional pathological tissue staining method are that the morphological structure and the number of nucleolus cannot be clearly shown, the nucleolus and nuclear plasma cannot be distinguished, and great troubles are brought to morphological examination and analysis of malignant tumor cells.

At present, the cytological detection of CTC is basically divided into two types, namely chemical staining and immunofluorescence staining, wherein the chemical staining and the blood cell detection are the same, the repeatability is poor, the sensitivity is low, and the morphological structure of nucleolus cannot be clearly shown basically; the existing immunofluorescence staining method focuses on displaying cell surface antigens, basic morphology of cells is displayed through DAPI lining staining to judge whether the cells are CTC cells, and particularly morphological structure and number of nucleoli of the CTC cells cannot be displayed. For example, the Cell Search CTC detection method approved by FDA in the united states (a representative staining inspection method at home and abroad, which is improved but cannot comprehensively reflect the authenticity of CTC), the criterion for CTC is: the cells are round or oval, EpCAM, CK and vimentin are positively stained, DAPI can see the form of cell nucleus, and CD45 is negatively stained, namely the CTC. Since cell surface antigens may change as cells undergo shedding, transfer and mutation processes, such as EMT or MET transformation, expression of certain antigens by cells changes, and certain blood resident cells express illegal editing of antigens, indicating a positive reaction of certain antigens staining. Therefore, the detection of CTC by the current universal immunofluorescence staining method at home and abroad is difficult to judge the CTC, and false positive or false negative can easily occur. The staining method of the invention makes up the defects of the staining method, and in addition to displaying the overall morphology of malignant cells (part of cytopathological characteristics of malignant cells with large nucleus, malformation and disordered nuclear-plasma ratio), the method is mainly characterized in that the key links and pathological change bases of the pathological morphological changes of malignant cells, such as nucleolar morphological structure, quantity and distribution of malignant cells, karyokaryokinesis and the like, can be clearly displayed. The method increases reliable analysis and identification basis for the detection personnel in distinguishing malignant cells. The reagent and the staining method are also suitable for the detection, analysis and identification of other body cavity cast-off cells and blood disease cells, and the basic principle is the same.

The detection of peripheral blood Circulating Tumor Cells (CTC) is an unknown reliable method for early cancer discovery and diagnosis, and the improvement of the sensitivity and specificity of detection is the basis for the popularization and use of the method. The traditional histopathological examination, exfoliative cytological examination and blood cell examination mainly use chemical staining for many years, can see the basic structures of malignant tumor cells, such as the morphological structures of cell membranes, cell plasma and cell nucleuses, and show part of cytopathological characteristics of large nuclei, nuclear malformation and nuclear plasma ratio disorder of the malignant tumor cells. The cell nucleolus is the key of cell growth, development, differentiation and proliferation, malignant tumor lesion is exactly the cell differentiation and development problem, and the cell nucleolus is the first pathological change in the process of malignant tumor onset and disease progression, so the pathological morphological change of the cell nucleus and the nucleolus should be emphasized in the pathological examination of malignant tumor cells, and the change of the cell nucleus, particularly the change of the nucleolus, is the pathological morphological basis for identifying malignant tumor cells.

Disclosure of Invention

The invention aims to provide a human exfoliated cell fluorescent staining method and a kit. The staining method and the staining kit can clearly and completely display the cell nucleus, particularly the nucleolus which can mark the cell nucleus, and clearly show the number, the shape, the volume size and the distribution of the nucleolus. The key characteristic of human cytopathology is analyzed, and the antigen of cytoplasm and cell membrane is displayed by immunofluorescence staining method, so that the full-form observation and analysis of human exfoliative cells can be carried out.

The basic principle of the invention is as follows:

the kit and the staining method are used for detecting malignant exfoliated cells, SYTO RNA Select green fluorescent dye is used for staining nucleolus and nucleus of the exfoliated cells to be green, nucleolus green of the cells is particularly concentrated under a fluorescent microscope, and nucleolus shape and nucleolus number are clear and visible; wherein the nucleus is pale green (less green than the nucleolus). The red fluorescent dye is labeled by specific monoclonal antibodies such as Vimentin (Vimentin), epithelial cell adhesion molecule (EpCAM), Cytokeratin (CK) and the like, the cell pulp and the cell surface of the exfoliated cells are subjected to fluorescent staining, and red fluorescence is presented under a fluorescent microscope.

The technical scheme adopted by the invention is as follows:

according to the first aspect of the invention, the immunofluorescent staining kit for the human exfoliated cells comprises SYTO RNA Select green fluorescent dye with special affinity with nucleolus RNA, cell membrane and cytoplasm antigen marker specific monoclonal antibody labeled red fluorescent dye, cell fixing liquid, cell sealing liquid, rinsing liquid, diluting liquid, and cell membrane and cell nucleus membrane permeation liquid.

Specifically, the specific monoclonal antibody labeled red fluorescent dye is one or more of a combination of monoclonal antibodies labeled red fluorescent dyes selected from Vimentin (Vimentin), epithelial cell adhesion molecule (EpCAM) and Cytokeratin (CK). The Vimentin (Vimentin) also included EMT or MET transformations. The CK (cytokine) specifically includes CK8, CK18, CK19, and the like.

In a specific case, the specific monoclonal antibody labeled red fluorescent dye is a CD45 monoclonal antibody labeled with red fluorescent dye.

According to a second aspect of the present invention, there is provided a method for fluorescent staining of human exfoliated cells, comprising the steps of:

(1) providing and/or preparing a human exfoliated cell fluorescent staining kit according to the first aspect of the present invention;

(2) preparing an antibody binding reaction solution: respectively diluting SYTO RNA Select green fluorescent dye, cell membrane and cytoplasm antigen marker specific monoclonal antibody labeled red fluorescent dye by using diluent to prepare antibody combined reaction liquid;

(3) preparing an exfoliated cell specimen detection sheet;

(4) fixing the cells with cell fixing liquid for 10-20 min; washing with rinsing liquid for 1-3 times;

(5) incubating the cell membrane and nuclear membrane permeation solution with the cell specimen detection sheet at 37 ℃ for 10-20 minutes, and removing the solution;

(6) sealing with cell sealing liquid for 10-20 min, and removing the liquid;

(7) adding the antibody binding reaction solution obtained in the step (2), dyeing for 30-60 minutes at 37 ℃, removing the liquid, and washing for 1-3 times by using a rinsing solution;

(8) and (5) drying to be detected.

In specific cases, the exfoliated cells include circulating tumor cells from solid tumors entering the blood, exfoliated cells from pleural and peritoneal effusions, cerebrospinal fluid cells, exfoliated cells from vaginal cervix, exfoliated cells from urethra, exfoliated cells from gastrointestinal tract, and blood disease cells.

The invention mainly uses fluorescent dye SYTO RNA Select with special affinity to cell nucleus, especially cell nucleolus RNA to dye the cell nucleolus and the cell nucleus to be green, combines monoclonal antibodies of epithelial cell antigens and the like (EpCAM, CK, vimentin and the like) to mark red fluorescent dye, and dyes cytoplasm and cell surface to be red. Thus, the morphological structure of malignant cells can be completely displayed under a fluorescence microscope, the nucleus and nucleus of the cell show green, and the cytoplasm and the cell surface show red. The nuclei showed blue color when combined with DAPI staining. The number of the nuclei of the malignant cells is greater than or equal to 3, and the malignant cells are different in morphology and size, which is the core and key part of the invention. The number of non-malignant cells is generally one (normal blood cells do not have nucleoli), no more than 2, and the morphology is more regular and the size is basically consistent.

The staining method and the staining reagent can clearly and completely display the cell morphology, particularly the nucleolus of the cell nucleus, and the quantity, the morphology, the volume and the distribution of the nucleolus can be clearly seen; displaying the morphology of the cell nucleus; showing the morphology of the cytoplasm and cell surface.

The key point and the technical core of the invention are that the nucleus, especially the nucleolus which can mark the nucleus, can be clearly and completely displayed, and the quantity, the shape, the volume and the distribution of the nucleolus can be clearly seen. The key characteristic of human cytopathology is analyzed, and the antigen of cytoplasm and cell membrane is displayed by immunofluorescence staining method, so that the full-form observation and analysis of human exfoliative cells can be carried out.

Drawings

FIG. 1 is a photograph showing the result of fluorescent staining of CTC cells in example 1.

FIG. 2 is a photograph showing the result of fluorescent staining of pleural effusion and ascites exfoliative cells in example 2.

FIG. 3 is a photograph showing the result of fluorescent staining of blood disease cells in example 3.

Detailed Description

The invention provides four combined reagents and an operation method:

first, first group of reagent combinations and embodiments

Providing a first combination of exfoliated cell fluorescent staining reagents, the method of operation comprising the steps of:

(1) providing and/or preparing a fluorescent detection reagent for the characteristic of the exfoliated cells, wherein the fluorescent detection reagent comprises cell fixing liquid, cell sealing liquid, rinsing liquid, diluent, liquid passing through cell membranes and nuclear membranes, fluorescent dye Syto RNA select green of cell nuclear kernel markers, and fluorescent markers of the cell membranes and the cytoplasm antigens, namely Vimentin (Vimentin) monoclonal antibody.

(2) Preparing a cell fluorescent staining solution: respectively diluting a Syto RNA select green fluorescent dye and a Vimentin (Vimentin) monoclonal antibody fluorescent marker by using a diluent to prepare a cell fluorescent staining solution;

(3) preparing a peripheral blood cell specimen detection sheet;

(4) fixing the cells with cell fixing liquid for 10-20 min; washing with rinsing liquid for 1-3 times;

(5) incubating the cell membrane and nuclear membrane permeation solution with the cell specimen detection sheet at 37 ℃ for 10-20 minutes, and removing the solution;

(6) sealing with cell sealing liquid for 10-20 min, and removing the liquid;

(7) adding the cell fluorescent staining solution in the step (2), staining for 30-60 minutes at 37 ℃, removing the liquid, and washing for 1-3 times by using a rinsing solution;

(8) and (5) drying to be detected.

Second and third group of reagent combinations and embodiments

Providing a second combination of exfoliated cell fluorescent staining reagents, the method of operation comprising the steps of:

(1) providing and/or preparing an immunofluorescence cytology detection reagent, which comprises a cell fixing solution, a cell sealing solution, a rinsing solution, a diluting solution, a liquid passing through cell membranes and nuclear membranes, a Syto RNA select green fluorescence staining solution, a cytoplasm and cell membrane marker epithelial cell adhesion molecule EpCAM and a CK monoclonal antibody red fluorescence marker.

(2) Preparing a cell fluorescent staining solution: respectively diluting Syto RNA select green, EpCAM marked by red fluorescence and CK monoclonal antibodies by using a diluent to form a cell fluorescent staining solution;

(3) preparing a peripheral blood desquamation cell specimen detection sheet;

(4) fixing the cells with cell fixing liquid for 10-20 min; washing with rinsing liquid for 1-3 times;

(5) incubating the cell membrane and nuclear membrane permeation solution with the cell specimen detection sheet at 37 ℃ for 10-20 minutes, and removing the solution;

(6) sealing with cell sealing liquid at 37 deg.C for 10-20 min, and removing liquid;

(7) adding the cell fluorescent staining solution in the step (2), staining for 30-60 minutes at 37 ℃, removing the liquid, and washing for 1-3 times by using a rinsing solution;

(8) and (5) drying to be detected.

Third and third group of reagent combinations and embodiments

Providing a third combination of exfoliated cell fluorescent staining reagents, the method of operation comprising the steps of:

(1) providing and/or preparing a peripheral blood cell fluorescence detection reagent which comprises cell fixing liquid, cell sealing liquid, rinsing liquid, diluent, cell membrane and nuclear membrane permeable liquid, a cell nucleolar marker fluorescent dye Syto RNA select green, cell membrane and cytoplasm markers CK8, CK18, CK19, EpCAM and a vimentin monoclonal antibody red fluorescent marker;

(2) preparing a cell fluorescent staining solution: diluting Syto RNA select green, red fluorescence-labeled CK (CK8, CK18 and CK19), EpCAM and monoclonal antibodies of vimentin by using a diluent to form a cell fluorescent staining solution;

(3) preparing an exfoliated cell specimen detection sheet;

(4) fixing the cells with cell fixing liquid for 10-20 min, and washing with rinsing liquid for 1-3 times;

(5) incubating the cell membrane and nuclear membrane permeation solution with the cell specimen detection sheet at 37 ℃ for 10-20 minutes, and removing the solution;

(6) sealing with cell sealing liquid for 10-20 min, and removing the liquid;

(7) adding the cell fluorescent staining solution in the step (2), staining for 30-60 minutes at 37 ℃, removing the liquid, and washing for 1-3 times by using a rinsing solution;

(8) and (5) drying to be detected.

Fourth and fourth group of reagent combinations and embodiments

Providing a fourth hematological disease cell (e.g., leukemia cell) fluorescent staining method in combination with the reagent, the method comprising the steps of:

(1) providing and/or preparing an exfoliated cell fluorescence detection reagent, which comprises cell fixing solution, cell sealing solution, rinsing solution, diluent, cell membrane and nuclear membrane permeable solution, a cell nucleolar marker fluorescent dye Syto RNA select green, a cell membrane and a cytoplasm marker CD45 monoclonal antibody red fluorescent marker;

(2) preparing a fluorescent dyeing reaction solution: diluting the Syto RNA select green and the CD45 monoclonal antibody marked by red fluorescence by using a diluent to form a fluorescent staining reaction solution;

(3) blood disease cell specimen detection sheet;

(4) fixing cells with cell fixing liquid for 10-20 min, and washing with rinsing liquid for 1-3 times;

(5) incubating the cell membrane and nuclear membrane permeation solution with the cell specimen detection sheet at 37 ℃ for 10-20 minutes, and removing the solution;

(6) sealing with cell sealing liquid for 10-20 min, and removing the liquid;

(7) adding the fluorescent dyeing reaction solution in the step (2), dyeing for 30-60 minutes at 37 ℃, removing the liquid, and rinsing for 1-3 times by using a rinsing solution;

(8) and drying in the air to be detected.

The following describes the present invention in detail by way of specific examples.

Example 1 fluorescent staining of CTC cells

(1) The reagent of the kit comprises:

cell fixing solution: methanol, acetone, and paraformaldehyde.

The cell sealing liquid comprises: PBS (containing 1% BSA or calf serum) at pH 7.2-7.4.

The rinsing liquid comprises: PBS (containing Tween-20) with pH value of 7.2-7.4.

The diluent comprises: PBS (containing 1% BSA or calf serum) at pH 7.2-7.4.

The cell membrane and cell nucleus membrane permeable liquid comprises: 0.2-0.5% triton X-100.

Nucleolar marker fluorescent dye Syto RNA select green.

CK. EpCAM, monoclonal antibody red fluorescent marker of vimentin.

Other reagents: phosphate Buffered Saline (PBS), calf serum albumin (BSA), donkey plasma (clear), Tween-20, Tris-HCl, etc.

(2) The fluorescent staining method comprises the following steps:

preparing CTC cell detection tablet.

② fixing the cells by methanol or 4 percent paraformaldehyde for 10-20 minutes.

③ washing the mixture for 1 to 3 times by using a rinsing solution (pH7.2 to 7.4PBS (containing Tween-20)).

Fourthly, 0.5 percent triton is used for incubation with the cell slice (37 ℃) for 10 to 20 minutes, and the liquid is discarded.

Blocking with 1% BSA for 10-20 min (37 deg.C), and removing the liquid.

Sixthly, adding a monoclonal antibody staining reagent of Syto RNA select green and red fluorescence labeling CK, EpCAM and vimentin, and staining for 30-60 minutes at 37 ℃. Discarding the clear liquid, and washing with rinsing liquid (pH7.2-7.4PB (containing Tween-20) for 1-3 times.

Seventhly, sealing, and performing microscopic examination (fluorescence confocal microscope or fluorescence microscope).

Referring to FIG. 1, in a fluorescence microscope, the CTC nucleoli appeared dark green and concentrated nucleoli, the nuclear plasma appeared light green, and the plasma and cell surface appeared red.

Example 2 fluorescent staining of exfoliated cells in pleural ascites

(1) Reagent kit reagent:

cell fixing solution: methanol, acetone, and paraformaldehyde.

The cell sealing liquid comprises: PBS (containing 1% BSA or calf serum) at pH 7.2-7.4.

The rinsing liquid comprises: PBS (containing Tween-20) with pH value of 7.2-7.4.

The diluent comprises: PBS (containing 1% BSA or calf serum) at pH 7.2-7.4.

The cell membrane and cell nucleus membrane permeable liquid comprises: 0.2-0.5% triton X-100.

Nucleolar marker fluorescent dye Syto RNA select green.

EpCAM, CK monoclonal antibody red fluorescence marker.

Other reagents: phosphate Buffered Saline (PBS), calf serum albumin (BSA), donkey plasma (clear), Tween-20, Tris-HCl, etc.

(2) The fluorescent staining method comprises the following steps:

preparing the detecting tablet of the exfoliated cells of the pleural effusion and the ascites.

② fixing the cells by methanol or 4 percent paraformaldehyde for 10-20 minutes.

③ washing the mixture for 1 to 3 times by using a rinsing solution (pH7.2 to 7.4PBS (containing Tween-20)).

Fourthly, 0.5 percent triton is used for incubation with the cell slice (37 ℃) for 10 to 20 minutes, and the liquid is discarded.

Blocking with 1% BSA for 10-20 min (37 deg.C), and removing the liquid.

Sixthly, adding a Syto RNA select green and a red fluorescence labeling EpCAM and CK monoclonal antibody staining reagent, and staining for 30-60 minutes at 37 ℃. The liquid was discarded and washed 1 to 3 times with a rinsing solution (pH7.2 to 7.4PBS (containing Tween-20)).

Seventhly, sealing, and performing microscopic examination (fluorescence confocal microscope or fluorescence microscope).

Referring to FIG. 2, under a fluorescence microscope, the nucleoli of the exfoliated malignant cells appeared dark green and concentrated nucleoli, the nuclear plasma appeared light green, and the cell plasma and cell surface appeared red.

Example 3 fluorescent staining of hematological disease cells

(1) Reagent kit

Cell fixing solution: methanol, acetone, and paraformaldehyde.

The cell sealing liquid comprises: PBS (containing 1% BSA or calf serum) at pH 7.2-7.4.

The rinsing liquid comprises: PBS (containing Tween-20) with pH value of 7.2-7.4.

The diluent comprises: PBS (containing 1% BSA or calf serum) at pH 7.2-7.4.

The cell membrane and cell nucleus membrane permeable liquid comprises: 0.2-0.5% triton X-100.

Nucleolar marker fluorescent dye Syto RNA select green.

The CD45 monoclonal antibody red fluorescent marker.

Other reagents: phosphate Buffered Saline (PBS), calf serum albumin (BSA), donkey plasma (clear), Tween-20, Tris-HCl, etc.

(2) Fluorescent staining method

Preparing a blood disease cell detection sheet.

② fixing the cells by methanol or 4 percent paraformaldehyde for 10-20 minutes.

③ washing the mixture for 1 to 3 times by using a rinsing solution (pH7.2 to 7.4PBS (containing Tween-20)).

Fourthly, 0.5 percent triton is used for incubation with the cell slice (37 ℃) for 10 to 20 minutes, and the liquid is discarded.

Blocking with 1% BSA for 10-20 min (37 deg.C), and removing the liquid.

Sixthly, adding a staining agent of the Syto RNA select green and a red fluorescence labeling CD45 monoclonal antibody, and staining for 30-60 minutes at 37 ℃. The liquid was discarded and washed 1 to 3 times with a rinsing solution (pH7.2 to 7.4PBS (containing Tween-20)).

Seventhly, sealing, and performing microscopic examination (fluorescence confocal microscope or fluorescence microscope).

Referring to FIG. 3, under a fluorescence microscope, the nucleoli of the hematological cells showed dark green color and concentrated nucleoli, the nuclear plasma showed light green color, and the cell plasma and cell surface showed red color.

Claims (5)

1. A kit for fluorescence staining human exfoliated cells is characterized by comprising SYTO RNA Select green fluorescent dye (RNA fluorescent dye) with special affinity with nucleolus RNA, red fluorescent dye marked by specific monoclonal antibodies of cell membrane and cytoplasm antigen markers, cell fixing liquid, cell sealing liquid, rinsing liquid, diluent, and liquid for communicating cell membranes with cell nuclear membranes.

2. The kit for immunofluorescent staining of exfoliated cells of a human body according to claim 1, wherein the specific monoclonal antibody labeled red fluorescent dye is one or a combination of more of monoclonal antibody labeled red fluorescent dyes selected from vimentin, cytokeratin and epithelial cell adhesion molecules.

3. The kit for immunofluorescent staining of human peripheral blood exfoliated cells according to claim 1, wherein the specific monoclonal antibody labeled red fluorescent dye is a CD45 monoclonal antibody labeled with red fluorescent dye.

4. An immunofluorescence staining method for human exfoliated cells is characterized by comprising the following steps:

(1) providing and/or preparing an immunofluorescent human exfoliated cell staining kit according to any one of claims 1 to 3;

(2) preparing a cell fluorescent staining solution: respectively diluting SYTO RNA Select green fluorescent dye, cell membrane and cytoplasm antigen marker specific monoclonal antibody labeled red fluorescent dye by using diluent to prepare cell fluorescent staining solution;

(3) preparing an exfoliated cell specimen detection sheet;

(4) fixing the cells with cell fixing liquid for 10-20 min; washing with rinsing liquid for 1-3 times;

(5) incubating the cell membrane and the cell nucleus membrane with the cell specimen detection sheet at 37 ℃ for 10-20 minutes, and removing the liquid;

(6) sealing with cell sealing liquid for 10-20 min, and removing the liquid;

(7) adding the cell fluorescence reaction solution in the step (2), dyeing for 30-60 minutes at 37 ℃, discarding the liquid, and washing for 1-3 times by using a rinsing solution;

(8) and (5) drying to be detected.

5. The immunofluorescent staining method for exfoliated cells of human bodies according to claim 4, wherein the exfoliated cells include circulating tumor cells of solid tumors entering the blood, exfoliated cells of pleural and peritoneal effusions, cerebrospinal fluid cells, exfoliated cells of vaginal cervix, exfoliated cells of urethra, exfoliated cells of gastrointestinal tract and blood disease cells.

Images