Cell immobilization transparent synergistic treatment reagent and application thereof

Abstract

The invention discloses a cell immobilization transparent synergistic treatment reagent, which is genipin with a concentration of not less than 10 mM. When the reagent is used for fixing cells, the effect of transparentizing the cells can be achieved, a transparent agent does not need to be additionally added during immune cell chemical detection, the detection efficiency can be improved, and meanwhile, as a fixing reagent, the reagent is high in penetrating power, can keep the inherent form and structure of the cells during survival, and is convenient to use. The effective binding activity of the antigen can be maintained by a stable original conformation, so that the staining intensity of immune cell chemistry is improved and enhanced. Meanwhile, compared with common chemical fixing agents such as aldehydes and alcohols, genipin is a natural biological cross-linking agent and has no volatile toxicity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a cell fixation transparent collaborative processing reagent and application thereof. Background Art

[0002] Immunocytochemistry is the application of the principle of antigen-antibody binding to detect the distribution of macromolecules such as polypeptides and proteins in cells. The key factor affecting the staining effect is fixation. The purpose of fixation is to keep the internal and external structures of the cell clear, maintain the integrity of the cell membrane, prevent the cell from autolysis after death, resulting in blurred sections, and prevent the cell from excessive shrinkage or expansion and losing its original shape; cross-link or coagulate the intracellular proteins to maintain the antigenicity of the cells. In summary, how to preserve cell samples and how to handle cells are the keys to the success of immunocytochemistry.

[0003] Chemical fixatives are generally divided into aldehyde fixatives, alcohol fixatives, mercury fixatives, oxidant fixatives, picrates, etc.

[0004] At present, the commonly used cell fixatives are mainly aldehydes (formaldehyde, glutaraldehyde and paraformaldehyde) and alcohols (ethanol). The principle of alcohol fixatives is coagulation fixation, that is, fixation by precipitating intracellular proteins and sugars; the advantages are strong penetration and good antigenicity preservation; the disadvantages are that the fixation effect on cell nuclei is not ideal, strong dehydration, easy to cause tissue shrinkage and hardening, affecting the quality of slices. The principle of aldehyde fixatives is cross-linking fixation, and the advantages are moderate activity, good penetration, moderate cross-linking degree, low tissue shrinkage rate, good morphological structure preservation, and can fix most biological macromolecules; the disadvantage is that most aldehydes have a pungent odor, which can be smelled at low concentrations, and have a stimulating effect on the skin and mucous membranes. In addition, the aldehyde group cross-links with the amino group of the antigen protein to form a carboxyl group, which causes spatial obstacles to the three-dimensional conformation of the antigenic determinant; the grid structure formed by intermolecular cross-linking may partially or completely cover some antigenic determinants, making them unable to be fully exposed, which may reduce antigenicity.

[0005] Cell transparency helps to reduce the scattering of different components within the cell, making the internal structure of the cell easier to observe and improving the clarity of observation. Commonly used transparent agents are xylene, acetone, etc. Under different circumstances, the transparent step takes from a few minutes to tens of minutes.

[0006] In summary, for immunocytochemistry experiments, it is very necessary to develop a new type of efficient cell fixation and transparent co-processing reagent that has strong penetration, is non-toxic, and does not affect the antigen conformation. Summary of the invention

[0007] In order to solve at least one of the above problems, the present invention provides a cell fixation and transparent co-processing reagent and application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a cell fixation and transparent co-processing reagent, wherein the reagent is genipin.

[0009] In some embodiments of the present invention, the concentration of genipin is not less than 10 mM.

[0010] The second aspect of the present invention provides the use of the cell fixation and transparency co-processing reagent described in the first aspect in the preparation of a cell sample suitable for tissue cell detection and analysis.

[0011] The third aspect of the present invention provides the use of the cell fixation and transparent co-processing reagent described in the first aspect in the preparation of a kit suitable for tissue cell detection and analysis.

[0012] In some specific embodiments of the present invention, the tissue cell detection and analysis is immunocytochemistry.

[0013] In some specific embodiments of the present invention, the use of genipin for cell fixation can enhance the staining intensity of immunocytochemistry and can be used to detect antigens located in the cell cytoplasm, cell nucleus, and cell membrane.

[0014] In some specific embodiments of the present invention, in the immunocytochemistry, after the cells are fixed with genipin in the cell fixation step, no cell clearing step is performed.

[0015] In some specific embodiments of the present invention, the concentration of genipin is not less than 10 mM. In some specific embodiments of the present application, preferably, the concentration of genipin is not less than 15 mM.

[0016] The fourth aspect of the present invention provides an immunocytochemical detection kit, characterized in that it comprises the cell fixation and transparent co-processing reagent described in the first aspect.

[0017] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a cell fixation transparent synergistic treatment reagent, which as a fixing reagent can react with carboxyl and amino groups at the same time, has strong penetration, can maintain the inherent morphology and structure of cells when they survive, and can also make the antigen maintain effective binding activity in a stable original conformation, thereby improving the staining intensity of enhanced immunocytochemistry. At the same time, compared with commonly used chemical fixatives such as aldehydes and alcohols, genipin is the product of geniposide extracted from gardenia fruit after hydrolysis by B-glucosidase, is an excellent natural biological cross-linking agent, has no volatile toxicity, and is safer to use. Its chemical structure contains an active cyclopentane group, has a certain chemical reaction activity, can react with a variety of functional groups, thereby achieving modification effects such as coupling of different molecules; compared with the classic fixation method composed of 10% neutral formalin, the cell staining intensity and positive rate fixed with genipin are better, and the reagent can also achieve the effect of making cells transparent while fixing the cells, and no additional transparent agent is required during immunocytochemistry detection, which can improve the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The cell staining results of experimental groups 1-6 in Example 1 of the present invention are shown; Figure 2 The cell staining results of experimental groups 7-8 and control groups 1-2 in Example 1 of the present invention are shown; Figure 3 The cell staining results of the experimental group and the control group in Example 2 of the present invention are shown; Figure 4 The cell staining results of the experimental group and the control group in Example 3 of the present invention are shown; DETAILED DESCRIPTION The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0020] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0021] Example 1 During conventional immunocytochemistry testing, after the cells are fixed with a fixative, they need to be treated to be transparent. Xylene is the most widely used tissue transparent agent. It can quickly dissolve organic substances on the cell membrane, quickly melt organic substances in the tissue, and absorb moisture in the tissue material, thereby making the tissue more transparent, and conveniently and effectively studying the cell structure. When genipin is used as a cell fixative, it has the effect of making the cells transparent, and there is no need to perform additional transparent treatment on the cells. In order to demonstrate the effect of genipin on cell transparency, this embodiment uses long-term cultured cervical cancer cells Hela as materials for experiments, and the selected target is Ki-67, and the subcellular localization is the nucleus.

[0022] 1. Experimental cells The Hela cells used in the present invention are from Guangzhou Geneo Biotechnology Co., Ltd.

[0023] 2. Cell culture reagents RPMI1640+10%FBS.

[0024] 3. Cultivate cells Hela cells were selected for routine cell recovery culture and cell passage. Cells in the logarithmic growth phase were collected and centrifuged at 120M, centrifuged at 1000rpm for 5min, the supernatant was discarded, and 10mL PBS was added for resuspending. The cells were centrifuged at 1000rpm for 5min, and the supernatant was discarded. The cells were washed 3 times in total. The cell samples resuspended in 10mL PBS were divided into 10 portions, each with about 30M cells. They were used as cell samples for experimental groups 1-8 and control groups 1-2.

[0025] 4. Cell Fixation (1) Preparation of cell fixative: Experimental group: Genipin solid powder was weighed and dissolved in PBS to make the final concentrations of 5 mM, 10 mM, 15 mM, and 30 mM.

[0026] Control group: Measure 100 ml of formaldehyde, weigh 4 g of sodium dihydrogen phosphate and 6.5 g of disodium hydrogen phosphate, add 900 ml of distilled water, mix well and prepare 10% neutral formalin.

[0027] (2) Cell fixation: The cells collected and grouped in step 3 were centrifuged at 1000 rpm for 5 min and the supernatant was discarded. The experimental group was divided into two groups, 1-4 and 5-8. Ten times the sedimentation volume of 5 mM, 10 mM, 15 mM, and 30 mM genipin were added to the four groups in each group, respectively. 10% neutral formalin was added to the control groups 1 and 2. The ten groups were fixed with shaking at 20 rpm at laboratory temperature for 16 h.

[0028] 5. Cell dehydration After fixation, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 5 mL PBS to resuspend the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash once; dehydrate the cell pellet with 75% ethanol, 95% ethanol, and anhydrous ethanol respectively, and let it stand for 60 min each time a gradient is changed.

[0029] 6. Cell transparency Experimental groups 1-4 and control group 1 were treated with xylene for transparency: after cell dehydration, the anhydrous ethanol was discarded, 15 mL of xylene was added to make the cell pellet transparent, and the pellet was allowed to stand at room temperature for 30 min.

[0030] Experimental groups 5-8 and control group 2 were not subjected to transparency treatment.

[0031] 7. Preparation of cell wax blocks Place the cell clumps in parallel in a metal embedding holder and soak them in wax for 3 to 4 hours to make cell wax blocks.

[0032] 8. Cell immunohistochemistry detection The wax blocks prepared in the experimental group and the control group were sliced, and the cell suspension was prepared according to the relevant methods described in the patent CN202011356471.8 A method for preparing a semi-solid cell suspension and its application, and the prepared cell suspension was subjected to immunohistochemical staining experiments. The immunohistochemical staining experiment was carried out using the LYNX480 fully automatic immunohistochemical staining machine of Tuling (Hangzhou) Biomedicine Co., Ltd. The supporting Ki-67 target primary antibody working solution (I10092-05) and auxiliary reagents (I20032C) were purchased from the commercial kit of Tuling (Hangzhou) Biomedicine Co., Ltd. The experiment adopted the conventional immunohistochemical experimental process, and the primary antibody working solution (I10092-05) was diluted once with PBS, and the rest was carried out according to the instructions of the kit.

[0033] 9. Results The experimental results of experimental group 1-8 and control group 1-2 are as follows Figure 1 and Figure 2 shown.

[0034] Results: From Figure 1 and Figure 2It can be seen that the cell nuclei, cytoplasm, and cell membranes of experimental groups 1-4, 6-8, and control group 1 are clearly visible, and the staining intensity of experimental groups 3-4 and 7-8 is close, and slightly stronger than control group 1, and significantly stronger than experimental groups 1-2 and 6; the staining of experimental group 5 and control group 2 is fuzzy and the positioning is unclear. This shows that 10mM genipin is used as a cell fixative in immunocytochemical detection experiments, which has a transparent effect, but the fixation effect of the cell nucleus localization target is not good; 15mM genipin is used as a cell fixative in immunocytochemical detection experiments, which can not only fix intracellular antigens well, but also has a transparent effect, and can simultaneously replace the two reagents of 10% neutral formalin (cell fixative) and xylene (transparent agent) in the traditional method. Therefore, when using genipin as a cell fixation transparent synergist for immunocytochemical experiments, there is no need to use an additional transparent agent, thereby improving the detection efficiency.

[0035] Example 2 HMB45 is encoded by the PMEL gene and is an important structural component of melanosomes, involved in the synthesis and storage of melanin. HMB45 is distributed in immature and activated melanocytes (such as inflammation, hyperplastic blood vessels or potential tumors), not in mature and quiescent melanocytes, and its subcellular localization is cytoplasm. In this example, the mouse melanoma cell line B16-F0 was selected as the detection cell to demonstrate the detection effect of genipin as a fixed transparent synergistic reagent on HMB45 in B16-F0 cells.

[0036] 1. Experimental cells The melanoma cell line B16-F0 used in the present invention is from Shanghai Baili Biotechnology Co., Ltd.

[0037] 2. Cell culture reagents RPMI1640+10%FBS.

[0038] 3. Cultivate cells B16-F0 cells were selected for routine cell recovery culture and cell passaging. Cells in the logarithmic growth phase were collected and centrifuged at 60M, 1000rpm for 5min, the supernatant was discarded, 10mL PBS was added for resuspending, 1000rpm for 5min, the supernatant was discarded, and the cells were washed 3 times in total. The cell samples resuspended in 10mL PBS were divided into 2 portions, each with about 30M cells.

[0039] 4. Cell Fixation (1) Preparation of cell fixative: Experimental group: Genipin solid powder was weighed and dissolved in PBS to a final concentration of 15 mM.

[0040] Control group: Measure 100 ml of formaldehyde, weigh 4 g of sodium dihydrogen phosphate and 6.5 g of disodium hydrogen phosphate, add 900 ml of distilled water, mix well and prepare 10% neutral formalin.

[0041] (2) Cell fixation: The cells collected and grouped in step 3 were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. 15 mM genipin (10 times the volume of the precipitate) was added to one sample, which was used as the experimental group; 10% neutral formalin was added to the other sample, which was used as the control group. The experimental and control groups were fixed by shaking at 20 rpm at room temperature for 16 h.

[0042] 5. Cell dehydration After fixation, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 5 mL PBS to resuspend the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash once; dehydrate the cell pellet with 75% ethanol, 95% ethanol, and anhydrous ethanol respectively, and let it stand for 60 min each time a gradient is changed.

[0043] 6. Cell transparency After the cell dehydration was completed, the anhydrous ethanol was discarded, and 15 mL of xylene was added to make the cell pellet of the control group transparent, and then the pellet was allowed to stand at room temperature for 30 min.

[0044] The experimental groups do not need to be transparent.

[0045] 7. Preparation of cell wax blocks Place the hardened and transparent cell clumps in parallel in a metal embedding seat and soak them in wax for 3 to 4 hours to make cell wax blocks.

[0046] 8. Cell immunohistochemistry detection The wax blocks prepared for the experimental group and the control group were sliced, and the cell suspension was prepared according to the relevant methods described in the patent CN202011356471.8 A method for preparing a semi-solid cell suspension and its application, and the prepared cell suspension was subjected to immunohistochemical staining experiments. The immunohistochemical staining experiment was carried out using the LYNX480 fully automatic immunohistochemical staining machine of Tuling (Hangzhou) Biomedicine Co., Ltd. The HMB45 target primary antibody working solution (I10162-05) and auxiliary reagents (I20032C) used in conjunction were purchased from the commercial kit of Tuling (Hangzhou) Biomedicine Co., Ltd. The experiment adopted the conventional immunohistochemical experimental process, and the primary antibody working solution (I10162-05) was diluted once with PBS, and the rest was carried out according to the instructions of the kit.

[0047] 9. Results The experimental results of the two groups are as follows: Figure 2 shown.

[0048] Results: From Figure 3 From the control group, it can be seen that after the cells were fixed and transparentized with 10% neutral formalin and xylene, the subcellular localization was cytoplasm, the staining intensity was medium, the positive rate was medium, the internal and external structures of the cells were clear, the cell membrane was intact, and there was no shrinkage. Figure 3 It can be seen from the experimental group that after using 15mM genipin for cell fixation and transparency, the subcellular localization is cytoplasm, the staining intensity is moderate to strong, the positive rate is high, the intracellular and extracellular structures are clear, the cell membrane is intact, and there is no shrinkage. This shows that genipin has a better effect on preserving the three-dimensional structure of cytoplasmic protein antigens than 10% neutral formalin during cell fixation. Therefore, when detecting antigens located in the cytoplasm, genipin can be used as a good cell fixative in immunocytochemistry experiments.

[0049] Example 3 Synaptophysin (SYP) is a membrane-integrated glycoprotein with a molecular weight of about 38 kDa, and its subcellular localization is the cell membrane. It is mainly expressed in the presynaptic membrane of neurons, adrenal medulla, pancreatic islet cells and other tissues. In this example, the retinoblastoma cell line Y79 was selected as the detection cell to demonstrate the detection effect of genipin as a fixed transparent synergistic reagent on SYP in Y79 cells.

[0050] 1. Experimental cells The Y79 cells used in the present invention are from Shanghai Baili Biotechnology Co., Ltd.

[0051] 2. Cell culture reagents RPMI1640+10%FBS.

[0052] 3. Cultivate cells Y79 cells were selected for routine cell recovery culture and cell passaging. Cells in the logarithmic growth phase were collected and centrifuged at 60M, 1000rpm for 5min, the supernatant was discarded, 10mL PBS was added for resuspending, 1000rpm for 5min, the supernatant was discarded, and the whole was washed 3 times. The cell sample after resuspending in 10mL PBS was divided into 2 portions, each with about 30M cells.

[0053] 4. Cell Fixation (1) Preparation of cell fixative: Experimental group: Genipin solid powder was weighed and dissolved in PBS to a final concentration of 10 mM.

[0054] Control group: Measure 100 ml of formaldehyde, weigh 4 g of sodium dihydrogen phosphate and 6.5 g of disodium hydrogen phosphate, add 900 ml of distilled water, mix well and prepare 10% neutral formalin.

[0055] (2) Cell fixation: The cells collected and grouped in step 3 were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. One group was added with 10 times the volume of the precipitate, and used as the experimental group; the other group was added with 10% neutral formalin, and used as the control group. The experimental and control groups were fixed by shaking at 20 rpm at room temperature for 16 h.

[0056] 5. Cell dehydration After fixation, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 5 mL PBS to resuspend the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash once; dehydrate the cell pellet with 75% ethanol, 95% ethanol, and anhydrous ethanol respectively, and let it stand for 60 min each time a gradient is changed.

[0057] 6. Cell transparency After the cell dehydration was completed, the anhydrous ethanol was discarded, and 15 mL of xylene was added to make the cell pellet of the control group transparent, and then the pellet was allowed to stand at room temperature for 30 min.

[0058] The experimental groups do not need to be transparent.

[0059] 7. Preparation of cell wax blocks Place the hardened and transparent cell clumps in parallel in a metal embedding seat and soak them in wax for 3 to 4 hours to make cell wax blocks.

[0060] 8. Cell immunohistochemistry detection The wax blocks prepared for the experimental group and the control group were sliced, and the cell suspension was prepared according to the relevant methods described in the patent CN202011356471.8 A method for preparing a semi-solid cell suspension and its application, and the prepared cell suspension was subjected to immunohistochemical staining experiments. The immunohistochemical staining experiment was carried out using the LYNX480 fully automatic immunohistochemical staining machine of Tuling (Hangzhou) Biomedicine Co., Ltd. The SYP target primary antibody working solution (I10412-05) and auxiliary reagents (I20032C) used in conjunction were purchased from the commercial kit of Tuling (Hangzhou) Biomedicine Co., Ltd. The experiment adopted the conventional immunohistochemical experimental process, and the primary antibody working solution (I10412-05) was diluted once with PBS, and the rest was carried out according to the instructions of the kit.

[0061] 9. Results The experimental results of the two groups are as follows: Figure 4 shown.

[0062] Results: From Figure 4 From the control group, it can be seen that after the cells were fixed and made transparent using 10% neutral formalin and xylene, the subcellular localization was the cell membrane, the staining intensity was medium, the positive rate was medium, the internal and external structures of the cells were clear, the cell membrane was intact, and there was no shrinkage. Figure 4It can be seen from the experimental group that after using 10mM genipin for cell fixation and transparency, the subcellular localization is the cell membrane, the staining intensity is moderate, the positive rate is high, the internal and external structures of the cell are clear, the cell membrane is intact, and there is no shrinkage. This shows that genipin preserves the three-dimensional structure of membrane protein antigens during cell fixation, which is equivalent to 10% neutral formalin. Therefore, when detecting antigens located on the cell membrane, genipin can also be used as a good cell fixative in immunocytochemistry experiments, and the concentration used can be reduced to 10mM.

[0063] The results of Examples 1 to 4 show that when detecting antigens located in the cell nucleus, cytoplasm, and cell membrane, the fixation effect of not less than 10 mM genipin is stronger than that of 10% neutral formalin, which can maintain the inherent morphology and structure of the cell during survival, and can also enable the antigen to maintain effective binding activity in a stable original conformation, thereby improving the staining intensity of immunocytochemistry. In addition, when using genipin as a cell fixation and transparency synergist for immunocytochemistry experiments, no additional transparency agent is required, which can improve the detection efficiency.

[0064] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A cell fixation and transparent co-processing reagent, characterized in that: The reagent is genipin.

2. The cell fixation and transparent co-processing reagent according to claim 1, characterized in that: The concentration of genipin is not less than 10 mM.

3. Use of the cell fixation and transparent synergistic processing reagent according to claim 1 or 2 in preparing cell samples suitable for tissue cell detection and analysis.

4. The use according to claim 3, characterized in that: In the cell sample preparation process, after genipin is used to fix the cells in the cell fixation step, the cell transparentization step is not performed.

5. Use of the cell fixation and transparent synergistic processing reagent according to claim 1 or 2 in the preparation of a kit suitable for tissue cell detection and analysis.

6. The use according to claim 5, characterized in that: The tissue cell detection and analysis is immunocytochemistry.

7. The use according to any one of claims 3 to 6, characterized in that: The concentration of genipin is not less than 10 mM.

8. An immunocytochemical detection kit, characterized in that: The invention comprises the cell fixation and transparent co-processing reagent according to claim 1 or 2.

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