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what is the process of Immunofluorescence?

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Immunofluorescence technique (Immunofluorescence technique, IF), also known as fluorescent antibody technique, is one of the earliest developed labeling immunological techniques. In 1941, Coons and others successfully used fluorescein to label antibodies for the first time and established immunofluorescence technology. After decades of development, the technology has become quite mature.

Immunofluorescence technique
Immunofluorescence technique

The method of using fluorescent antibody to trace or check the corresponding antigen is called the fluorescent antibody method; the method of using known fluorescent antigen markers to trace or check the corresponding antibody is called the fluorescent antigen method. These two methods are collectively referred to as immunofluorescence technology, because fluorescent pigments can not only bind to antibody globulins to detect or locate various antigens, but also bind to other proteins to detect or locate antibodies, but in actual work, fluorescent antigens Technology is rarely applied, so people are used to calling it fluorescent antibody technology, or immunofluorescence technology, and fluorescent antibody methods are more commonly used. Using immunofluorescence technology to display and inspect antigens or hapten substances in cells or tissues is called immunofluorescence cell (or tissue) chemistry technology.

Immunofluorescence experiment process:

1. Sample preparation:

For adherent cell samples, during subculture, inoculate the cells into a petri dish with 70% ethanol-treated coverslips, and after the cells grow into a single layer, take out the coverslips and wash them twice with PBS; for suspension growth Take the logarithmic growth of the cell sample and wash it twice with PBS at 1000 rpm for 5 min. Use a cytocentrifugal shaker to prepare cell sheets or directly prepare cell smears.

2. Fixed

Choose an appropriate fixative to fix the cells according to your needs. The fixative includes: organic solvents (methanol, ethanol, acetone, etc.), cross-linking agents (4% PFA, 10% neutral formalin), and formaldehyde is the most widely used, but It is not applicable to phosphorylated antibodies, which will result in the transfer of phosphoprotein from the membrane surface to the cytoplasm. Ice anhydrous methanol or anhydrous ethanol should be selected. The fixation time depends on the size and type of the sample. Generally, 18-24h is ideal. The cell fixation time is shorter, and 2% formaldehyde can be used for 20 minutes at room temperature.


Permeabilization is to allow the antibody to enter the cell. 0.5% TritonX-100 can be permeated for 20 minutes at room temperature (intracellular antigen, cell membrane antigen is skipped). In addition, acetone can also be used as a permeabilizing agent.


The slides were immersed in PBS 3 times, 3min/time, blotted dry with absorbent paper, and goat serum was dripped on the slides, and blocked at room temperature for 30 minutes. Commonly used blocking solutions include: serum from the same source as the secondary antibody, BSA or goat serum.

5.Antibody incubation

Dilute the primary antibody with the primary antibody diluent according to the appropriate ratio, absorb the blocking solution with absorbent paper, add the diluted primary antibody dropwise to each slide and place it in a humid box, incubate overnight at 4°C, recover the primary antibody, and add PBST. Wash slowly on a shaker for 5 minutes, and wash 3 times. Operate in the dark, absorb the washing solution and add the fluorescent secondary antibody dropwise, incubate in a humid box for 1 hour, recover the secondary antibody, wash with PBST for 5 minutes, and wash 3 times.

6.Counterstain the nucleus

DAPI was added dropwise and incubated for 5 min in the dark. The specimens were stained with nucleus, and then washed with PBST for 5 min, and washed 3 times to remove excess DAPI.

7.Mounting and observation

Aspirate the liquid, mount the slide with a mounting solution containing anti-fluorescence quencher, and observe under a fluorescence microscope.

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