Principle and steps of immunofluorescence staining
Immunofluorescence is also known as immunofluorescent antibody. The principle of immunofluorescence experiments is to label fluorescein on the corresponding antibody and directly react with the corresponding antigen. The method has the advantages of simple method, high specificity, less non-specific fluorescent staining, and relatively large amount of labeled antibody. Localization of antigenic material in tissues or cells using antigen-antibody reactions.
The immunofluorescence staining steps are detailed below:
1. Add 0.01 mol/L, pH 7.4 PBS to the specimen to be tested, and discard it after 10 minutes to keep the specimen at a certain humidity.
2. Drip the appropriately diluted fluorescently labeled antibody solution to cover the specimen completely and place it in a covered enamel box for a certain period of time (Reference: 30 min).
3. Remove the slide and place it on the slide holder. Rinse with PBS of 0.01mol/L, pH7.4, then soak in PBS three cylinders of 0.01mol/L and pH7.4 in sequence, 3-5 per cylinder. Min, oscillation from time to time.
4. Remove the slide and use a filter paper to remove excess water, but do not dry the specimen. Add a drop of buffered glycerin and cover with a coverslip.
5. Immediately observe with a fluorescence microscope. Observe the specific fluorescence intensity of the specimen, generally indicated by "+": (-) no fluorescence; (±) very weak suspicious fluorescence; (+) weak fluorescence, but clearly visible; (++) fluorescent bright; (+ ++--++++) Fluorescent. The specific fluorescence intensity of the specimen to be tested is "++" or more, and the various controls are (±) or (-), which can be judged as positive.
For a more complete understanding of immunofluorescence markers, you can browse the Solaibao experiment.
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