Cell immobilization method for immunofluorescent staining

Abstract

The invention discloses a cell immobilization method for immunofluorescent staining, which comprises the following steps: (1) placing a culture solution of cells to be immobilized in a cell culture pore plate, absorbing and discarding 45-55% of the culture solution, adding and absorbing and discarding a precooled 4% tissue cell immobilization solution with the same volume as the culture solution, and immobilizing at room temperature for 13-17 minutes; (2) absorbing and discarding all liquid in the pore plate, adding and absorbing and discarding precooled 4% tissue cell fixing liquid with the same volume, and fixing at room temperature for 13-17 minutes; and (3) absorbing and abandoning the stationary liquid, washing with a PBS (Phosphate Buffer Solution), and then carrying out immunofluorescence staining. According to the cell fixation method for immunofluorescence staining, the normal structure of cells can be effectively maintained, the number of cells during immunofluorescence is large, cell morphological change caused by cell shedding can be prevented, the fluorescence effect of immunofluorescence detection is high, and a fluorescence image is attractive and accurate.

Description

Technical Field

[0001] The invention relates to a cell fixing method for immunofluorescence staining, belonging to the technical field of immunofluorescence. Background Art

[0002] Immunofluorescence technique, also known as fluorescent antibody technique, is the earliest developed technique in labeled immunology. It is also a technique established on the basis of immunology, biochemistry and microscopy. Immunofluorescence cytochemistry is based on the principle of antigen-antibody reaction. First, the known antigen or antibody is labeled with fluorescein to make a fluorescent marker, and then the fluorescent antibody (or antigen) is used as a molecular probe to examine the corresponding antigen (or antibody) in cells or tissues, and finally the nature and location of the antigen or antibody are determined, and the content is measured using quantitative technology.

[0003] The purpose of fixation is to preserve the tissue and cell structure intact, terminate the activity of intracellular enzymes and other metabolic activities, provide the true situation at a certain time point that the researcher wants to study, and in the subsequent staining, no too dark background will be produced, thereby ensuring the reliability and accuracy of the experiment. This is also the first step in sample pretreatment in immunofluorescence staining experiments. Fixation is divided into dry fixation and wet fixation, and wet fixation is currently the most classic fixation method. Traditional fixation methods are not effective in fixing cells, and are prone to cause cell morphology changes and cell detachment. Therefore, it is necessary to optimize the fixation method. Most of the existing research focuses on optimizing the composition of the fixative to achieve the purpose of maintaining the original morphology of the cells, and there are few specific methods for fixing cells. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a cell fixation method for immunofluorescence staining.

[0005] The present invention is achieved through the following technical solutions:

[0006] A cell fixation method for immunofluorescence staining comprises the following steps:

[0007] (1) Place the culture medium of the cells to be fixed in a cell culture well plate, aspirate 45% to 55% (volume percentage) of the culture medium, add and aspirate the same volume of pre-cooled 4% tissue cell fixative, and fix at room temperature for 13 to 17 minutes;

[0008] (2) Aspirate all liquid in the well plate, add an equal volume of pre-cooled 4% tissue cell fixative, and fix at room temperature for 13 to 17 minutes;

[0009] (3) Aspirate the fixative and wash with PBS buffer before proceeding to the next step of immunofluorescence staining.

[0010] Furthermore, in step (1), 50% of the culture solution is discarded.

[0011] Furthermore, in steps (1) and (2), precooling refers to precooling at 4°C.

[0012] Furthermore, in the steps (1) and (2), the fixed time is 15 minutes.

[0013] Furthermore, the cells to be fixed are selected from neuronal cells, and the immunofluorescence staining is performed on neuron-specific markers TUJ1 or DCX.

[0014] The cell fixation method for immunofluorescence staining of the present invention can effectively maintain the normal structure of cells, increase the number of cells during immunofluorescence, and prevent cell morphological changes caused by cell shedding. The fluorescence effect of immunofluorescence detection is high, and the fluorescence image is beautiful and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Pictures of cells under a microscope before fixation, after fixation, and after TUJ1 immunofluorescence staining (comparative example).

[0016] Figure 2 : Microscopic images of cells before fixation, after fixation, and after DCX immunofluorescence staining (comparative example).

[0017] Figure 3 : Pictures of cells under a microscope before fixation, after fixation, and after TUJ1 immunofluorescence staining (Example).

[0018] Figure 4 : Microscopic images of cells before fixation, after fixation, and after DCX immunofluorescence staining (Example). DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0020] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods already available in the prior art.

[0021] The 4% tissue cell fixative used in the present invention is a commonly used fixative in the prior art and can be purchased on the market, for example, from Bensheng (Tianjin) Health Technology Co., Ltd.

[0022] Comparative Example Traditional Cell Fixation Method

[0023] Here are the steps:

[0024] (1) After the neuronal cells are cultured, white light images are taken under a microscope;

[0025] (2) Aspirate all the old culture medium from the plate and add an equal volume of 4% tissue cell fixative precooled at 4°C and fix at room temperature for 15 min.

[0026] (3) The fixative solution was discarded, the cells were washed with PBS buffer, and then immunofluorescence staining of neuron-specific markers TUJ1 and DCX was performed.

[0027] The pictures of cells under the microscope before and after fixation and TUJ1 immunofluorescence staining are as follows: Figure 1 As shown; the pictures of cells under the microscope before fixation, after fixation, and after DCX immunofluorescence staining are as follows Figure 2 shown.

[0028] Example Cell fixation method of the present invention

[0029] Here are the steps:

[0030] (1) After the neuronal cells are cultured, white light images are taken under a microscope;

[0031] (2) Aspirate half of the old culture medium in the well plate and add an equal volume of 4% tissue cell fixative precooled at 4°C and fix at room temperature for 15 min.

[0032] (3) Aspirate all the liquid in the well plate, add and discard the same volume of 4% tissue cell fixative precooled at 4°C, and fix at room temperature for 15 min;

[0033] (4) The fixative was discarded, the cells were washed with PBS, and then immunofluorescence staining of neuron-specific markers TUJ1 and DCX was performed.

[0034] The pictures of cells under the microscope before and after fixation and TUJ1 immunofluorescence staining are as follows: Figure 3 As shown; the pictures of cells under the microscope before fixation, after fixation, and after DCX immunofluorescence staining are as follows Figure 4 shown.

[0035] contrast Figure 1 to Figure 4It can be seen that, by adopting the cell fixing method of the present invention, there is no obvious difference in the number of cells before and after fixation, the typical morphological structure of neurons is maintained, and the positioning is accurate after immunofluorescence staining; while in the control group, the number of cells before and after fixation is obviously reduced, a large number of neurons fall off, and the neurons that do not fall off lose the typical morphological structure of neurons, affecting the authenticity and accuracy of the experimental results, and after immunofluorescence staining, non-specific staining is easily produced, and the fluorescence effect of immunofluorescence detection is low, which affects the beauty of the fluorescence image.

[0036] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A cell fixation method for immunofluorescence staining, characterized in that: The following steps are involved: (1) Place the culture medium of the cells to be fixed in a cell culture well plate, aspirate 45% to 55% of the culture medium, add and aspirate the same volume of pre-cooled 4% tissue cell fixative, and fix at room temperature for 13 to 17 minutes; (2) Aspirate all liquid in the well plate, add an equal volume of pre-cooled 4% tissue cell fixative, and fix at room temperature for 13 to 17 minutes; (3) Aspirate the fixative, wash with PBS buffer, and then perform immunofluorescence staining.

2. The cell fixation method for immunofluorescence staining according to claim 1, characterized in that: In the step (1), 50% of the culture solution is discarded.

3. The cell fixation method for immunofluorescence staining according to claim 1, characterized in that: In the steps (1) and (2), precooling refers to precooling at 4°C.

4. The cell fixation method for immunofluorescence staining according to claim 1, characterized in that: In the steps (1) and (2), the fixed time is 15 minutes.

5. The cell fixation method for immunofluorescence staining according to claim 1, characterized in that: The cells to be fixed are selected from neuronal cells, and the immunofluorescence staining is performed on neuron-specific markers TUJ1 or DCX.

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