Natural environment-friendly stationary liquid

Abstract

The invention discloses a natural environment-friendly stationary liquid. The component of the stationary liquid is honey. The invention also provides a method for fixing cells and preparing a cell wax block based on the stationary liquid. Compared with 10% formalin, the fixing effect of the fixing liquid provided by the invention is equivalent, and the fixing liquid provided by the invention is environment-friendly and pollution-free; and the cell paraffin block prepared by using the stationary liquid is verified by an immunohistochemical method, the cell morphological structure is clear, and the dyeing positioning is accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of cell biology, and in particular relates to a natural and environmentally friendly fixing liquid. Background Art

[0002] Formalin Fixed Paraffin Embedded (FFPE) samples are a common method for long-term preservation of pathological samples in clinical practice. Pre-fixation of samples is the first and important step in tissue processing. The main purpose of fixation is to maintain the vital state of the tissue, prevent bacterial decay, prevent autolysis, and increase the refractive index of the tissue. Formalin has been a commonly used fixative in tissue pathology processing because of its ease of use, economic feasibility, and fast fixation speed.

[0003] However, formaldehyde is a pathogenic irritant to the eyes, nose, and oral mucosa. Repeated exposure or prolonged inhalation in an occupational environment poses a potential health hazard. Non-formalin fixatives do not contain aldehyde components, thus avoiding any potential toxic effects. Currently, there is an aldehyde-free fixative on the market, but the effect is inferior to that of 10% formalin. Therefore, we tried to find an alternative that is environmentally friendly, safe, and has a good fixation effect. Summary of the invention

[0004] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a natural and environmentally friendly fixing liquid.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A first aspect of the present invention provides a natural and environmentally friendly fixing liquid.

[0007] Furthermore, the fixing solution contains honey.

[0008] Furthermore, the concentration of the honey is 3% to 10%.

[0009] Preferably, the concentration of the honey is 5% to 8%.

[0010] More preferably, the concentration of honey is 5%.

[0011] Suitable fixatives do not damage antigenicity, and can also maintain the structure of tissues and cells and allow macromolecules to enter cells and bind to antigens. The main fixative groups classified according to the mechanism of action include aldehydes, alcohols, oxidants, mercurials and picrates, and the aldehydes include C1-C6 chain alkyl or C1-C8 alkylene dialdehydes, and the alcohols include ethylene glycol, propylene glycol, glycerol, sorbitol and mannitol.

[0012] In one embodiment, the fixative is formaldehyde, preferably 10% neutral buffered formalin (NBF). However, formaldehyde is a pathogenic irritant to the eyes, nose, and oral mucosa. Repeated contact or long-term inhalation in an occupational environment poses a potential health hazard. Therefore, the inventors of the present application sought an environmentally friendly, safe, and effective fixative substitute, and found that the fixative effect of honey is comparable to that of 10% formalin. However, not all concentrations of honey have a fixative effect. Preferably, 3% to 10% honey has a fixative effect, 5% to 8% is the best, and most preferably, 5% honey has the best effect.

[0013] A second aspect of the present invention provides a product.

[0014] Furthermore, the product comprises the fixing liquid described in the first aspect of the present invention.

[0015] Furthermore, the product also includes conventional reagents in the process of preparing cell wax blocks.

[0016] Furthermore, conventional reagents used in the cell wax block preparation process include ethanol and xylene.

[0017] Preferably, the product further comprises instructions.

[0018] Preferably, the kit further comprises any one or more of n-butanol, methanol, isopropanol, acetone, 2-ethylhexyl benzoate, 2-ethylhexyl palmitate, 2-ethylhexyl coconut oil, 2-ethylhexyl stearate, 2-ethylhexyl acetate, benzyl benzoate, polyethylene glycol methyl ether methacrylate, tetrahydroxypropyl ethylenediamine solution, and limonene.

[0019] A third aspect of the present invention provides a cell fixation method.

[0020] Furthermore, the cell fixation method comprises adding the fixative solution described in the first aspect of the present invention to the cell sample to be fixed.

[0021] Furthermore, the volume ratio of the fixative solution to the cell sample to be fixed described in the first aspect of the present invention is 5:1-10:1.

[0022] As is well known in the art, the main function of the fixative is to fix the cell structure and maintain the integrity of the cell DNA and protein. In order to allow the fixative to diffuse into the tissue sample and make it as close as possible to the living tissue structure, the cells need to be immersed in a large amount of fixative. The volume of the fixative is at least 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more that of the cells. In a specific embodiment of the present invention, the volume of the fixative is 10 times that of the cells.

[0023] In the present invention, the fixing step is performed at a changed temperature or at room temperature.

[0024] Preferably, the changing temperature refers to treating the reagents alternately with hot and cold, for example, first treating at a low temperature of <15° C. and then at a high temperature of >20° C. In a low temperature environment, the fixative is easy to penetrate into the cells, and a high temperature can increase the cross-linking kinetics.

[0025] Preferably, when fixation is performed at room temperature, the duration of the fixation treatment may be 12-48 hours, preferably about 24 hours.

[0026] A fourth aspect of the present invention provides a method for preparing a cell wax block.

[0027] Furthermore, the method comprises fixing the cell sample to be fixed using the cell fixation method described in the first aspect of the present invention.

[0028] Furthermore, the method also includes the steps of dehydration, transparency, wax impregnation and embedding.

[0029] In some embodiments, the cell sample to be fixed is a digested cell, and the method of digesting cells is conventionally known to those skilled in the art.

[0030] Specifically, the reagent used for the digestion includes one or a combination of proteinase K, trypsin, collagenase A, deoxyribonuclease, hyaluronidase, and pronase. In a specific embodiment of the present invention, trypsin is used for digestion.

[0031] Furthermore, the steps of dehydration, transparency, wax infiltration and embedding are conventionally known to those skilled in the art.

[0032] Preferably, the dehydration can be performed in a pressurized environment or a normal atmospheric pressure environment.

[0033] Preferably, the mold used in preparing the cell wax block can be of any shape, and commonly used molds in the art include cubes and cuboids.

[0034] Preferably, the method further comprises a step of coagulating the cell sample with a coagulating agent to fix it after fixation and before dehydration.

[0035] Preferably, the coagulant comprises one or more of xanthan gum, sodium alginate, pectin, propylene glycol alginate, methylcellulose, gum arabic, guar gum, polysaccharide, and gelatin. In a specific embodiment of the present invention, the coagulant is a polysaccharide.

[0036] Preferably, the method further comprises the step of freezing the wax block and then cutting it into wax slices of suitable thickness.

[0037] Preferably, the cells on any of the 1st to 1500th slices obtained by the slicing have complete structures and are evenly distributed, and can be used as quality control cell wax slices for identifying the quality of diagnostic products and as quality control products.

[0038] Preferably, the method provided by the present invention is applicable to any cells, including cell lines or cells isolated from cancer patient samples.

[0039] Specifically, the samples include: peripheral blood, blood, serum, plasma, urine, saliva, semen, milk, cerebrospinal fluid, tears, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder washing fluid and bronchoalveolar lavage fluid or tissue (including clinical tissue).

[0040] Specifically, the cell line may be a cancer cell line, a self-prepared cancer cell line with a specific genotype, or a commercially available tumor cell line.

[0041] The cancer described in the present invention includes blood cancer: acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma; solid cancer: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, malignancy), pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, and CNS tumors (e.g., glioma, astrocytoma, medulloblastoma, craniopharyogioma), ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0042] The cancer cell lines of the present invention exemplarily include: breast cancer: 4T1 (mouse breast cancer cells), ZR-75-1 (human breast cancer cells), ZR-75-30 (human breast cancer cells), Bcap-37 (human breast cancer cells), BC-009 (human breast cancer cells), BC-019 (human breast cancer cells), BC-020 (human breast cancer cells), BC-021 (human breast cancer cells), BC-022 (human breast cancer cells), MA-891 (mouse breast cancer highly metastatic cells), MCF-7 (human breast cancer cells); lung cancer: 95-D (human highly metastatic lung cancer cells), PLA-801 D (human highly metastatic lung cancer cells), A-427 (human lung cancer cells), Calu-1 (human lung cancer cells), Calu-3 (human lung adenocarcinoma cells (pleural effusion)), H1395 (human lung adenocarcinoma cells), H1975 (human lung adenocarcinoma cells), H460 (human large cell lung cancer cells), H661 (human large cell lung cancer cells), LA795 (mouse lung adenocarcinoma cells), HCC827 (human non-small cell lung cancer cells), H157 (human non-small cell lung adenocarcinoma cells); liver cancer: BEL-7402 (human liver cancer cells), BEL-7404 (human liver cancer cells), H-4-II-E-C3 (rat liver cancer cells cells), HB611 (human hepatoma cells), HepB1.2 (human hepatoma cells), HHCC (human hepatoma cells), H-97 (human highly metastatic hepatoma cells), HCCLM3 (human highly metastatic hepatoma cells), HCCC-9810 (human cholangiocarcinoma cells), Hep3B2.1-7 (human hepatoma cells), BEL-7405 (human hepatoma cells); blood cancer: 6T-CEM (human T cell leukemia cells), TF-1 (human blood leukemia cells), THP-1 (human monocytic leukemia), L6565 (mouse leukemia clone cell line); kidney cancer: OS-RC-2 (human kidney cancer Cells), A-498 (human renal cancer cells), G-401 (human renal carcinoma Wilms cells), SW839 (human renal cancer cells); ovarian cancer: A2780 (human ovarian cancer cells), Anglne (human ovarian cancer cells), Caov-3 (human papillary ovarian adenocarcinoma cells), CoC1 (human ovarian cancer cells), CoC1 / DDP (human ovarian cancer cells CoC1 cisplatin-resistant substrain), ES-2 (human ovarian clear cell carcinoma cells); 5637 (human bladder cancer cells); 22RV1 (human prostate cancer cells); A-431 (human epidermal cancer cells); Acc-2 (human salivary gland adenoid cystic carcinoma cells). In the specific embodiments of the present invention, MCF-7 cell line (Pnosai) is used as an example for verification.

[0043] Specifically, those skilled in the art can determine the number of cells collected in the present invention according to the specific purpose of use. Preferably, 1-10×10 7 (1×107 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 ,7×10 7 , 8×10 7 ,9×10 7 , 10×10 7 ) are all appropriate cell numbers.

[0044] A fifth aspect of the present invention provides a cell wax block.

[0045] Furthermore, the cell wax block is prepared according to the preparation method described in the fourth aspect of the present invention.

[0046] The sixth aspect of the present invention provides any one of the following applications.

[0047] Furthermore, the application includes:

[0048] Use of the fixing solution described in the first aspect of the present invention in preparing cell wax blocks;

[0049] The use of the cell wax block prepared by the preparation method described in the fourth aspect of the present invention in the preparation of disease diagnosis products.

[0050] Furthermore, the diagnostic products include detection kits or detection equipment based on NGS, FISH, IHC, and immunogenomics detection methods.

[0051] Preferably, the diagnostic product is applicable to any one or a combination of the aforementioned cancers of the present invention.

[0052] Advantages and beneficial effects of the present invention: The present invention provides a natural and environmentally friendly fixative, which has a fixation effect comparable to that of 10% formalin, and the fixative provided by the present invention is environmentally friendly and pollution-free; and the use of the fixative to prepare cell wax blocks has been verified by immunohistochemistry methods, and the cell morphology and structure are clear and the staining positioning is accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shows the fixation effect of different concentrations of honey; Figure 2 Shows the comparison of fixation effect between 5% honey and fixatives on the market. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0055] Example

[0056] 1. Experimental Materials

[0057] The experimental materials and sources used in the examples of the present invention are shown in Table 1.

[0058] Table 1 Sources of experimental materials

[0059] MCF7 cells Punosa CL-0149 Anhydrous ethanol Tianjin Fuyu paraffin Shitai 80200-0009 Glass slides Japanese Matsunami PRO-03 Cytokeratin 8 & 18 Antibody Reagents (Immunohistochemistry) Zhongshan ZM-0315 10% formalin fixative Solebao G2161 4% paraformaldehyde fixative Solebao P1110 5% honey solution 10% sucrose solution Aldehyde-free fixative Harbin Green GAS-01

[0060] 2. Experimental Methods

[0061] MCF cells were collected and fixed for 24 h using 10% formalin, aldehyde-free fixative, 10% sucrose, 4% paraformaldehyde, 1% honey, 3% honey, 5% honey, 8% honey, 10% honey, 30% honey, and 50% honey, respectively, and then dehydrated and embedded to prepare cell wax blocks. CK8 / 18 immunohistochemistry was performed on each group of wax blocks to observe the integrity of cell structure and staining localization.

[0062] The specific experimental steps are as follows:

[0063] Cell block preparation:

[0064] 1. Cell culture, trypsin digestion until cells fall off, and collect cell pellets;

[0065] 2. Add 10 times the volume of 10% formalin / formaldehyde-free fixative / 10% sucrose / 4% paraformaldehyde / 1% honey / 3% honey / 5% honey / 8% honey / 10% honey / 30% honey / 50% honey fixative, vortex to mix, and fix at room temperature for 24 hours;

[0066] 3. Add polysaccharide solution to the fixed cells;

[0067] 4. Dehydration in a dehydrator;

[0068] 70% ethanol for 2 hours; 80% ethanol for 2 hours; 95% ethanol for 1 hour; 95% ethanol for 1 hour; 100% ethanol for 1 hour; 100% ethanol for 1 hour; ethanol:xylene (1:1) for 30 minutes; xylene for 30 minutes; xylene for 30 minutes; paraffin for 1 hour; paraffin for 1 hour; paraffin for 1 hour;

[0069] 5. Turn on the embedding machine, embed the dehydrated cells, and prepare them into cell wax blocks.

[0070] Specific steps of IHC experiment:

[0071] 1. Place the slices in 60℃-65℃ and bake for 2-3 hours;

[0072] 2. Dewaxing in xylene I for 20 min;

[0073] 3. Dewaxing in xylene II for 20 min;

[0074] 4. Anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol were treated for 2 min each;

[0075] 5. Soak in running tap water for a few seconds, then in distilled water for 2 minutes;

[0076] 6. Soak in PBS for 10 minutes;

[0077] 7. Antigen repair: refer to the instructions of the primary antibody for antigen repair;

[0078] 8. After returning to room temperature, soak in PBS;

[0079] 9. Treat with endogenous peroxidase blocking solution for 10 min and wash with deionized water;

[0080] 10. 10% goat serum, blocked in a 37°C wet box for 30 min;

[0081] 11. Incubate with primary antibody, refer to the instructions of the primary antibody;

[0082] 12. Incubate with secondary antibody, refer to the instructions of the secondary antibody;

[0083] 13. DAB staining for 10 minutes, pour out the DAB, and soak in tap water for 10 seconds;

[0084] 14. Hematoxylin staining for 2 minutes;

[0085] 15. Soak in running tap water for a few seconds, then rinse with distilled water;

[0086] 16. Differentiation in differentiation solution for 10-20 seconds;

[0087] 17. Soak in running tap water for a few seconds, then rinse with distilled water;

[0088] 18. Treat with blueing solution for 10-20s;

[0089] 19. Soak in running tap water for a few seconds, then rinse with distilled water;

[0090] 20. Dehydrate with gradient alcohol, make transparent with xylene, seal with neutral gum and observe.

[0091] 3. Experimental Results

[0092] IHC verification results (CK8 / 18) after fixation with different concentrations of honey solution Figure 1 As shown, it can be seen that not all concentrations of honey have a fixation effect. 3%~10% honey has a fixation effect, 5%~8% is better, and 5% honey has the best fixation effect.

[0093] The fixation effect of 5% honey was compared with that of the fixatives currently on the market. Figure 2 As shown, the fixation effect is found to be: 10% formalin > 5% honey > 4% paraformaldehyde > aldehyde-free fixative > 10% sucrose. This shows that the fixation effect of 5% honey is second only to formalin, comparable to formalin, and environmentally friendly and pollution-free, making it an excellent substitute for formalin. It also has a better fixation effect than paraformaldehyde and aldehyde-free environmentally friendly fixatives on the market.

[0094] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A natural environmentally friendly fixing liquid, characterized in that: The fixative comprises honey.

2. The fixing liquid according to claim 1, characterized in that The concentration of the honey is 3% to 10%; Preferably, the concentration of the honey is 5% to 8%; More preferably, the concentration of honey is 5%.

3. A product, characterized in that: The product comprises the fixing solution according to any one of claims 1-2.

4. The product according to claim 3, characterized in that The product also includes conventional reagents in the process of preparing cell wax blocks; Preferably, the conventional reagents used in the cell wax block preparation process include ethanol and xylene; Preferably, the product further comprises instructions.

5. A cell fixation method, characterized in that: The cell fixation method comprises adding the fixative solution according to any one of claims 1 to 2 to a cell sample to be fixed.

6. The cell fixation method according to claim 5, characterized in that: The volume ratio of the fixative according to any one of claims 1 to 2 to the cell sample to be fixed is 5:1 to 10:

1.

7. A method for preparing a cell wax block, characterized in that: The method comprises fixing the cell sample to be fixed by using the cell fixation method according to any one of claims 5 to 6.

8. The preparation method according to claim 7, characterized in that: The method further comprises the steps of dehydration, transparency, wax impregnation and embedding.

9. A cell wax block, characterized in that: The cell wax block is prepared according to the preparation method according to any one of claims 7-8.

10. Any of the following applications, characterized in that: The applications include: Use of the fixative according to any one of claims 1 to 2 in preparing cell wax blocks; Use of the cell wax block prepared by the preparation method according to any one of claims 7 to 8 in the preparation of disease diagnosis products; Preferably, the diagnostic product comprises a detection kit or detection equipment based on NGS, FISH, IHC, or immunohistochemistry detection methods.

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