Liquefying agent with cell membrane punching function and preparation method

Abstract

The invention relates to a liquefying agent with a cell membrane punching function and a preparation method, belongs to the technical field of biology, and is used for solving the problem that the existing liquefying agent cannot realize simultaneous implementation of cell punching and lysis effects. The liquefying agent with the cell membrane punching function is prepared from the following components in percentage by mass: 0.1 to 0.5 percent of acetylcysteine, 0.05 to 0.5 percent of polyethylene glycol octylphenol ether, 0.1 to 0.6 percent of nonionic polyacrylamide, 0.8 to 0.95 percent of sodium chloride, 0.01 to 0.05 percent of citric acid and the balance of water. The liquefying agent can be used for cracking mucus and punching a cell membrane at the same time, so that a coloring agent can smoothly pass through micropores of the cell membrane and directly enter the interior of the cell so as to realize a good dyeing function, the liquefying agent can synchronously realize two functions of cracking mucus and punching the cell membrane, and the cracking function is good; the cell morphology is complete, and when the immune cells are chemically stained, the antibody can directly enter the cells to realize a good antigen-antibody reaction.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a liquefier with a cell membrane punching function and a preparation method thereof. Background Art

[0002] In the field of pathology, immunocytochemistry and other staining methods directly using cells in fresh sputum are common and effective auxiliary diagnostic methods. However, the cell membrane has an obstructive effect on the dye, making it difficult or partially for the dye components to enter the cell to achieve the staining function. It is an effective solution to use technical means to make micropores in the cell membrane to allow antibodies to enter the cell smoothly.

[0003] However, current technologies have limitations. For example, existing mucus lysing solutions, sputum sample liquefaction agents and other similar products only have the function of lysing mucus, but lack the function of cell membrane perforation. Existing cell membrane perforators can only be used alone, and the application steps are cumbersome. In the prior art, cell membrane perforators cannot be directly added to liquefaction agents. This is because doing so will directly cause the components to produce chemical reactions and lose their original functions, or destroy the reaction system of the original cell preservation solution, affecting the function of lysing mucus. Summary of the invention

[0004] In view of the above analysis, the present invention aims to provide a liquefier with cell membrane perforation function, a preparation method and a staining method, so as to solve the problem that the existing liquefier cannot achieve cell perforation and lysis at the same time.

[0005] In the first aspect, the present invention provides a liquefier with a cell membrane perforating function. The liquefier comprises, in terms of mass percentage, 0.1-0.5% acetylcysteine, 0.05-0.5% polyethylene glycol octylphenyl ether, 0.1-0.6% non-ionic polyacrylamide, 0.8-0.95% sodium chloride, 0.01-0.05% citric acid, and the remainder is water.

[0006] Furthermore, the liquefier composition includes: 0.3% acetylcysteine, 0.3% polyethylene glycol octylphenyl ether, 0.5% non-ionic polyacrylamide, 0.9% sodium chloride, 0.02% citric acid, and the balance is water.

[0007] Furthermore, the water is distilled water.

[0008] In a second aspect, the present invention provides a method for preparing the liquefier having a cell membrane perforating function, wherein the method comprises the following steps:

[0009] (1) Weigh and set aside the raw materials according to the ratio of each raw material;

[0010] (2) Stir and dissolve the spare non-ionic polyacrylamide in water until a colorless and transparent solution is formed;

[0011] (3) Completely dissolve sodium chloride and citric acid in water, then add acetylcysteine, stir and dissolve it, and then add polyethylene glycol octyl phenyl ether to obtain a mixed solution;

[0012] (4) Add the said mixed solution to the said colorless and transparent solution to obtain the said liquefying agent.

[0013] Furthermore, the mass ratio of water in step (2) to water in step (3) is 6 - 8:2 - 4.

[0014] In the third aspect, the present invention provides a method for dyeing using the said liquefying agent, including the following steps:

[0015] (a) Add the cell specimen to the said liquefying agent, centrifuge and prepare a slide, add it to 95% ethanol for fixation to obtain a sample, and store it in PBS buffer solution;

[0016] (b) Add the H2O2 solution to the sample stored in PBS buffer solution, incubate at room temperature, add the primary antibody for incubation, wash, add the HRP-labeled secondary antibody for incubation, add the chromogenic solution, and the staining is completed.

[0017] Furthermore, in step (a), the volume ratio of the cell specimen to the said liquefying agent is 4 - 6:9 - 11.

[0018] Furthermore, in step (a), fix with 95% ethanol for 5 - 10 min.

[0019] Furthermore, in step (b), incubate at room temperature for 5 - 10 min.

[0020] Furthermore, in step (b), add the primary antibody and incubate at room temperature for 1 - 2 h.

[0021] Compared with the prior art, the present invention can at least achieve one of the following beneficial effects:

[0022] (1) The liquefying agent of the present invention can punch holes in the cell membrane while lysing mucus, enabling the staining agent to smoothly pass through the micropores of the cell membrane and directly enter the cell interior, thereby achieving the goal of good staining function, greatly simplifying the operation steps, and greatly improving the work efficiency; the liquefying agent of the present invention synchronously realizes the two functions of lysing mucus and punching holes in the cell membrane, and has good lysing function and complete cell morphology. During immunocytochemical staining, the antibody can directly enter the cell to achieve a good antigen-antibody reaction.

[0023] (2) Through a large number of experiments, the present invention has found that the polyacrylamide solution can be regarded as a network structure, and the mechanical entanglement and hydrogen bonds between the chains jointly form network nodes. These node-like solution structures can precisely provide effective protection for N-acetylcysteine molecules. In addition, the present invention has also found that after mixing polyethylene glycol octyl phenyl ether with the non-ionic polyacrylamide solution, it will combine with the node structure in the polyacrylamide solution, preventing the reaction with N-acetylcysteine, and thus achieving the effect that the two can exist in the same solution without affecting each other.

[0024] (3) When the cell sample is immersed in the liqueefying agent of the present invention, mucus liquefaction and cell membrane perforation can be completed synchronously, effectively avoiding the problem of loss of active ingredients caused by the need for multi-step sample transfer in the traditional method, and improving the diagnostic rate. Especially for samples with extremely small amounts of active ingredients, each transfer means a loss of the sample. Since the present invention does not require transfer, it can greatly reduce the loss of active ingredients. The liqueefying agent of the present invention simplifies the product form, and the transportation, storage, preservation, and use processes are all simplified, thereby improving efficiency and achieving better social benefits.

[0025] In the present invention, the above technical solutions can also be combined with each other to achieve more preferred combined solutions. Other features and advantages of the present invention will be described in the subsequent specification, and some advantages can be made obvious from the specification, or understood by implementing the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the content specifically pointed out in the specification and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings are only for the purpose of showing specific embodiments and are not considered to be a limitation of the present invention. Throughout the drawings, the same reference signs represent the same components.

[0027] Figure 1 It is a staining result diagram of the liqueefying agent of Example 1 in Application Example 1 of the present invention;

[0028] Figure 2 It is a staining result diagram of the liqueefying agent of Example 2 in Application Example 1 of the present invention;

[0029] Figure 3 It is a staining result diagram of the liqueefying agent of Example 3 in Application Example 1 of the present invention;

[0030] Figure 4 It is a staining result diagram of the liqueefying agent of Example 4 in Application Example 1 of the present invention;

[0031] Figure 5 It is a staining result diagram of the liqueefying agent of Example 5 in Application Example 1 of the present invention;

[0032] Figure 6It is the dyeing result diagram of the liquefier in Comparative Example 1 of Application Example 1 in the present invention;

[0033] Figure 7 It is the dyeing result diagram of the liquefier in Comparative Example 2 of Application Example 1 in the present invention;

[0034] Figure 8 It is the dyeing result diagram of the liquefier in Comparative Example 3 of Application Example 1 in the present invention;

[0035] Figure 9 It is the dyeing result diagram of the liquefier in Comparative Example 4 of Application Example 1 in the present invention;

[0036] Figure 10 It is the dyeing result diagram of the liquefier in Comparative Example 5 of Application Example 1 in the present invention;

[0037] Figure 11 It is the dyeing result diagram of the liquefier in Comparative Example 6 of Application Example 1 in the present invention;

[0038] Figure 12 It is the dyeing result diagram of the liquefier in the control group of Application Example 1 in the present invention;

[0039] Figure 13 It is the dyeing result diagram of the liquefier in Example 1 of Application Example 4 in the present invention;

[0040] Figure 14 It is the dyeing result diagram of the liquefier in Example 2 of Application Example 4 in the present invention;

[0041] Figure 15 It is the dyeing result diagram of the liquefier in Example 3 of Application Example 4 in the present invention;

[0042] Figure 16 It is the dyeing result diagram of the liquefier in Example 4 of Application Example 4 in the present invention;

[0043] Figure 17 It is the dyeing result diagram of the liquefier in Example 5 of Application Example 4 in the present invention;

[0044] Figure 18 It is the dyeing result diagram of the liquefier in Comparative Example 1 of Application Example 4 in the present invention;

[0045] Figure 19 It is the dyeing result diagram of the liquefier in Comparative Example 2 of Application Example 4 in the present invention;

[0046] Figure 20 It is the dyeing result diagram of the liquefier in Comparative Example 3 of Application Example 4 in the present invention;

[0047] Figure 21 It is the dyeing result diagram of the liquefier in Comparative Example 4 of Application Example 4 in the present invention;

[0048] Figure 22 This is the staining result diagram of the liquefying agent in Comparative Example 5 of Application Example 4 in the present invention;

[0049] Figure 23 This is the staining result diagram of the liquefying agent in Comparative Example 6 of Application Example 4 in the present invention;

[0050] Figure 24 This is the staining result diagram of the liquefying agent in the control group of Application Example 4 in the present invention. Detailed implementation manners

[0051] The following will specifically describe the preferred embodiments of the present invention in conjunction with the accompanying drawings, where the accompanying drawings form a part of the present invention and are used together with the embodiments of the present invention to illustrate the principle of the present invention, and are not used to limit the scope of the present invention.

[0052] A specific embodiment of the present invention discloses a liquefying agent with a cell membrane perforation function. By mass percentage, the composition of the liquefying agent includes: acetylcysteine: 0.1 - 0.5% (for example, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%), polyethylene glycol octyl phenyl ether: 0.05 - 0.5% (for example, 0.1%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%), non-ionic polyacrylamide: 0.1 - 0.6% (for example, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%), sodium chloride: 0.8 - 0.95% (for example, 0.82%, 0.84%, 0.86%, 0.88%, 0.90%, 0.92%, 0.94%), citric acid: 0.01 - 0.05% (for example, 0.015%, 0.020%, 0.025%, 0.030%, 0.035%, 0.040%, 0.045%), and the balance is water.

[0053] Specifically, the water is distilled water.

[0054] Preferably, acetylcysteine is 0.3%, polyethylene glycol octyl phenyl ether: 0.3%, non-ionic polyacrylamide: 0.5%, sodium chloride: 0.9%, citric acid: 0.02%, and the balance is water.

[0055] Compared with the prior art, the liquefying agent of the present invention can punch holes in the cell membrane while lysing mucus, enabling the staining agent to smoothly pass through the micropores of the cell membrane and directly enter the interior of the cell, thereby achieving the goal of a good staining function, greatly simplifying the operation steps, and greatly improving the work efficiency; the liquefying agent of the present invention synchronously realizes the two functions of lysing mucus and punching holes in the cell membrane, and has a good lysing function with intact cell morphology. During immunocytochemical staining, antibodies can directly enter the cells to achieve a good antigen-antibody reaction.

[0056] The cell sample is immersed in the liquefying agent of the present invention, and mucus liquefaction and cell membrane punching can be completed synchronously, effectively avoiding the problem of loss of active ingredients caused by multi-step sample transfer in the traditional method, and improving the diagnostic rate. Especially for samples with extremely small amounts of active ingredients, each transfer means a loss of the sample. Since the present invention does not require transfer, the loss of active ingredients can be greatly reduced. The liquefying agent of the present invention simplifies the product form, and the processes of transportation, warehousing, preservation, and use are all simplified, thereby improving efficiency and achieving better social benefits.

[0057] Acetylcysteine is an organic compound that cannot be directly mixed with polyethyleneglycol octylphenyl ether in the same solution. At room temperature, most of them will undergo chemical reactions, resulting in the destruction of the molecular structure and the loss of the original function.

[0058] Through a large number of experiments, the present invention found that the polyacrylamide solution can be regarded as a network structure, and the mechanical entanglement and hydrogen bonds between the chains jointly form network nodes. These node-like solution structures can precisely effectively protect the acetylcysteine molecules. In the present invention, non-ionic polyacrylamide is selected because through a large number of experiments, it is found that anionic or cationic polyacrylamide is likely to cause the agglomeration phenomenon of the sample. In addition, the present invention also found that after polyethyleneglycol octylphenyl ether is mixed with the non-ionic polyacrylamide solution, it will combine with the node structure in the polyacrylamide solution, preventing the reaction with acetylcysteine, and thus achieving the effect that the two exist in the same solution without affecting each other.

[0059] Another specific embodiment of the present invention discloses a preparation method of the above-mentioned liquefying agent with the function of punching holes in the cell membrane, including the following steps:

[0060] (1) Weigh and set aside according to the ratio of each raw material;

[0061] (2) Stir and dissolve the reserved non-ionic polyacrylamide in water until a colorless and transparent solution is formed;

[0062] (3) Completely dissolve sodium chloride and citric acid in water, then add acetylcysteine, stir and dissolve, and then add polyethyleneglycol octylphenyl ether to obtain a mixed solution;

[0063] (4) Add the said mixed solution to the said colorless and transparent solution to obtain the said liquefying agent.

[0064] Specifically, the mass ratio of water in step (2) to water in step (3) is 6 - 8:2 - 4. Preferably, the mass ratio is 7:3.

[0065] It should be noted that the cells in the present invention can be cells in sputum, cells in pleural effusion, or other cells that require simultaneous perforation and lysis.

[0066] Another specific embodiment of the present invention discloses a method for staining using the said liquefying agent, which includes the following steps:

[0067] (a) Add the cell specimen to the said liquefying agent, centrifuge and prepare a slide, add it to ethanol for fixation to obtain a sample, and store it in PBS buffer solution;

[0068] (b) Add the H2O2 solution to the sample stored in PBS buffer solution, incubate at room temperature, add the primary antibody for incubation, wash, add the HRP-labeled secondary antibody for incubation, add the chromogenic solution, and the staining is completed.

[0069] Specifically, in step (a), the volume ratio of the cell specimen to the said liquefying agent is 4 - 6:9 - 11.

[0070] Specifically, in step (a), the volume fraction of the ethanol is 95%. 。

[0071] Specifically, in step (a), fix with ethanol for 5 - 10 min, for example, 6 min, 7 min, 8 min, 9 min.

[0072] Specifically, in step (b), the mass fraction of H2O2 in the H2O2 solution is 3%.

[0073] Specifically, in step (b), incubate at room temperature for 5 - 10 min, for example, 6 min, 7 min, 8 min, 9 min.

[0074] Specifically, in step (b), add the primary antibody and incubate at room temperature for 1 - 2 h, for example, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h.

[0075] The primary antibody in the present invention is a commercially available raw material, and the dropping concentration is based on the recommended concentration in the instruction manual.

[0076] Specifically, in step (b), rinse 2 - 3 times with PBS or TBS buffer solution for 5 minutes each time to thoroughly remove the unbound antibody.

[0077] Specifically, in step (b), add the HRP-labeled secondary antibody and incubate at room temperature for 30 - 60 min, for example, 35 min, 40 min, 45 min, 50 min, 55 min.

[0078] Preferably, the HRP-labeled secondary antibody is anti-rabbit IgG or anti-mouse IgG. The addition amount is based on the recommended concentration in the instruction manual.

[0079] Specifically, the chromogenic solution is DAB chromogenic solution.

[0080] The following further explains the technical solution of the present invention in combination with specific embodiments.

[0081] The manufacturers and models of some raw materials in the following examples are as follows:

[0082] The manufacturers of acetylcysteine, polyethylene glycol octyl phenyl ether, and non-ionic polyacrylamide are all Sinopharm Chemical Reagent Co., Ltd., and they are all analytical pure reagents.

[0083] The manufacturer of the primary antibody CK5 / 6 is Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., and the model is OTI1F8.

[0084] The manufacturer of the primary antibody WT1 is Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., and the model is EP122.

[0085] The secondary antibody anti-rabbit IgG and the DAB chromogenic solution are different components in a universal kit (mouse / rabbit polymer detection system), and the manufacturer is Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., and the model is PV-6000D.

[0086] Example 1

[0087] A liquefying agent with cell membrane perforation function in this example, by mass percentage, the composition of the liquefying agent is shown in Table 1.

[0088] The preparation method of the liquefying agent with cell membrane perforation function in this example includes the following steps:

[0089] (1) Weigh and reserve according to the ratio of each raw material;

[0090] (2) Dissolve the reserved non-ionic polyacrylamide in distilled water with stirring until a colorless and transparent solution is formed;

[0091] (3) Completely dissolve sodium chloride and citric acid in distilled water, then add acetylcysteine, stir to dissolve, and then add polyethylene glycol octyl phenyl ether to obtain a mixed solution;

[0092] Among them, the mass ratio of water in step (2) to water in step (3) is 7:3;

[0093] (4) Add the said mixed solution to the said colorless and transparent solution to obtain the said liquefier.

[0094] Example 1-1

[0095] The composition of a liquefier with cell membrane perforation function in this example is the same as that in Example 1, except that the mass ratio of water in step (2) to water in step (3) is 6:4.

[0096] Example 1-2

[0097] The composition of a liquefier with cell membrane perforation function in this example is the same as that in Example 1, except that the mass ratio of water in step (2) to water in step (3) is 8:2.

[0098] Examples 2-5

[0099] The composition and ratio of the liquefier in Examples 2-5 are shown in Table 1, and the preparation method is the same as that in Example 1.

[0100] Comparative Examples 1-6

[0101] The composition and ratio of the liquefier in Comparative Examples 1-6 are shown in Table 1, and the preparation method is the same as that in Example 1.

[0102] Table 1

[0103]

[0104] Application Example 1

[0105] Use the liquefiers prepared in Example 1, Examples 2-5 and Comparative Examples 1-6 respectively, and select phosphate buffer (PBS) as the control group for immunocytochemical staining. In this application example, the cell sample is selected as sputum sample, and fresh sputum specimens submitted for clinical examination are selected to verify the exclusive treatment efficacy of the liquefier of the present invention on sputum samples. Each specimen is divided into 12 parts.

[0106] The specific staining method is as follows:

[0107] (a) Add 5 mL of sputum specimen to 10 mL of the said liquefier, centrifuge and prepare a slide, fix it with 95% ethanol for 10 min, and store it in PBS buffer to obtain a mixed solution;

[0108] (b) Add 3% hydrogen peroxide solution by mass fraction dropwise to the sample preserved in PBS buffer, ensuring complete coverage of the sample. Incubate at room temperature for 10 min, add primary antibody CK5 / 6 and incubate at room temperature for 1.5 h. Wash 3 times with PBS buffer, 5 min each time. Then add anti-rabbit IgG and incubate at room temperature for 45 min. Add DAB chromogenic solution and monitor under a microscope. The chromogenic time is 30 seconds to 5 min. Counterstain the cell nuclei with hematoxylin for 1 min, then clear and mount the slides to complete the staining.

[0109] The staining results of Example 1, Examples 2-5, Comparative Examples 1-6 and the control group are as Figure 1-12 shown.

[0110] CK5 / 6 is a marker of squamous epithelial cells, and the positive signal is located in the cytoplasm. After immunocytochemical staining of the specimens treated in Examples 1-5, as Figure 1-5 shown, the cell quantity is abundant, the cell morphology is intact, the cytoplasm of the vast majority of squamous cells shows brownish yellow, the staining positive rate is high, the staining intensity is strong, and the background is clear, showing a good staining effect, indicating that the liquefying agent of the present invention not only has good cell membrane penetration ability, but also has good efficacy for the stability of cell morphology and the enrichment of cell and other formed components. After immunocytochemical staining of the specimens treated in Comparative Examples 1-6, as Figure 6-11 shown, the cell quantity is scarce, the cell morphology is good, and the staining positive rate and staining intensity are not as good as Figure 1-5 . It shows that only the liquefying agent prepared with the components and ratios of the present invention has a better staining effect. After immunocytochemical staining of the specimens treated in the control group, as Figure 12 shown, the cell quantity is small, the cell morphology is good, the staining positive rate is lower, the staining intensity is weaker, and the staining effect is poor.

[0111] Application Example 2

[0112] The staining method in this application example is the same as that in Application Example 1, except that in step (a), 4 mL of sputum specimen is added to 9 mL of the liquefying agent and fixed with ethanol for 7.5 min. In step (b), incubate at room temperature for 5 min, add primary antibody CK5 / 6 and incubate at room temperature for 1 h.

[0113] The tests of the application example were carried out, and the results were basically the same. Due to limited space, they are not listed one by one.

[0114] Application Example 3

[0115] The staining method in this application example is the same as that in Application Example 1, except that in step (a), 6 mL of sputum specimen is added to 11 mL of the liquefying agent and fixed with ethanol for 5 min. In step (b), incubate at room temperature for 7.5 min, add primary antibody CK5 / 6 and incubate at room temperature for 2 h.

[0116] In this application example, tests of application examples were conducted, and the results were basically the same. Due to limited space, they will not be listed one by one.

[0117] Application Example 4

[0118] To verify the general processing efficiency of the liquefying agent of the present invention for other exfoliative cytology samples, the staining method in this application example is the same as that in Application Example 1. The difference is that the sputum sample is replaced with a fresh pleural effusion sample, and WT1 is selected as the primary antibody. The staining results of Examples 1-5, Comparative Examples 1-6, and the control group are as Figure 13-24 shown.

[0119] WT1 is a marker of mesothelial cells, and the positive signal is located in the cell nucleus. After the specimens treated in Example 1 and Examples 2-5 are subjected to immunocytochemical staining, as Figure 13-17 shown, the number of cells is abundant, the cell morphology is intact, the nuclei of mesothelial cells show brownish-yellow, lymphocytes are not stained, the staining positive rate is high, the staining intensity is strong, and the background is clear, showing a good staining effect, indicating that the liquefying agent of the present invention not only has good cell membrane penetration ability, but also has good efficiency for the stability of cell morphology and the enrichment of cell and other formed components. After the specimens treated in Comparative Examples 1-6 are subjected to immunocytochemical staining, as Figure 18-23 shown, the number of cells is abundant, the cell morphology is good, the nuclei of mesothelial cells show brownish-yellow, but non-specific cytoplasmic staining can be seen in some lymphocytes, and the overall staining effect is not good. It shows that only the liquefying agent prepared with the components and ratios of the present invention has a better staining effect. After the specimens treated in the control group are subjected to immunocytochemical staining, as Figure 24 shown, the number of cells is abundant, the cell morphology is good, only a small part of mesothelial cells show weak brownish-yellow, lymphocytes are not stained, the staining positive rate is low, the staining intensity is weaker, and the staining effect is poor.

[0120] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. A liquefying agent with the function of perforating cell membranes, characterized in that, By mass percentage, the composition of the liquefying agent includes: acetylcysteine: 0.1 - 0.5%, octylphenoxypolyethoxyethanol: 0.05 - 0.5%, nonionic polyacrylamide: 0.1 - 0.6%, sodium chloride: 0.8 - 0.95%, citric acid: 0.01 - 0.05%, and the balance is water.

2. The liquefier with the function of punching holes in cell membranes according to claim 1, wherein, The composition of the liquefying agent includes: acetylcysteine 0.3%, octylphenoxypolyethoxyethanol: 0.3%, nonionic polyacrylamide: 0.5%, sodium chloride: 0.9%, citric acid: 0.02%, and the balance is water.

3. A liquefier with a cell membrane perforation function according to claim 1 or 2, characterized in that, The water is distilled water.

4. A method for preparing a liquefying agent with cell membrane perforation function according to any one of claims 1-3, characterized in that, The method includes the following steps: (1) Weigh and reserve according to the ratio of each raw material; (2) Stir and dissolve the reserved nonionic polyacrylamide in water until a colorless and transparent solution is formed; (3) Completely dissolve sodium chloride and citric acid in water, then add acetylcysteine, stir and dissolve, and then add octylphenoxypolyethoxyethanol to obtain a mixed solution; (4) Add the mixed solution to the colorless and transparent solution to obtain the liquefying agent.

5. The preparation method of the liquefying agent with cell membrane perforating function according to claim 4, characterized in that, The mass ratio of water in step (2) to water in step (3) is 6 - 8:2 - 4.

6. A method for dyeing using the liquefying agent according to any one of claims 1-3, characterized in that, It includes the following steps: (a) Add the cell specimen to the liquefying agent, centrifuge and prepare a slide, add it to 95% ethanol for fixation to obtain a sample, and store it in PBS buffer; (b) Add the H2O2 solution to the sample stored in PBS buffer, incubate at room temperature, add the primary antibody for incubation, wash, add the HRP-labeled secondary antibody for incubation, add the chromogenic solution, and the staining is completed.

7. The method for dyeing according to the liquefying agent as claimed in claim 6, characterized in that, In step (a), the volume ratio of the cell specimen to the liquefying agent is 4 - 6:9 - 11.

8. The method for dyeing according to the liquefying agent as claimed in claim 6, characterized in that, In step (a), fix with 95% ethanol for 5 - 10 min.

9. The method for dyeing according to the liquefying agent as claimed in claim 6, characterized in that, In step (b), incubate at room temperature for 5 - 10 min.

10. The method for dyeing according to the liquefying agent as claimed in claim 6, characterized in that, In step (b), add the primary antibody and incubate at room temperature for 1 - 2 h.

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