1. The basic procedure for the preparation of immunofluorescent specimens is the same as the immunohistochemistry of DAB color development.
1. Immunofluorescence does not need to be treated with hydrogen peroxide, blocking and primary antibody incubation are the same as others.
2. Immunofluorescent secondary antibodies are incubated with different fluorescently labeled secondary antibodies, and the incubation time is determined according to the working concentration of the antibody.
3. After incubating the secondary antibody, the film can be patched, mounted, and observed after sufficient washing.
4. For immunofluorescence, use a special mounting tablet or glycerin: 0.01MPBS (1: 1) for mounting.
5. If conditions permit, you can purchase anti-quenching reagents and add them to the sealed tablets, and the specimens can be kept longer.
6. The incubation of fluorescent antibodies and subsequent treatment require light shut-off.
7. There may be many false positives for fluorescent antibody staining, and it is necessary to set positive and negative controls.
2. Matters needing attention
1. After fluorescence staining, the observation is usually completed within 1h, or stored at 4 ℃ for 4h. If the time is too long, the fluorescence will be weakened.
2. The following three controls need to be set for each test:
(1) Positive control: positive serum + fluorescent marker
(2) Negative control: negative serum + fluorescent marker
(3) Fluorescent marker control: PBS + fluorescent marker
3. The experience of immunofluorescence double labeling
1. Select primary antibodies from two different animals, use antibodies derived from rabbit and rat, secondary antibodies are labeled with different fluorescent signals, use donkey anti-rabbit-FITC (green) and donkey anti-rat- Tex-Red (red).
2. My approach is to incubate two primary antibodies at the same time, and then incubate two secondary antibodies at the same time. The antibody concentration and incubation time should be carefully explored. I feel that it is better to incubate primary antibodies overnight at 4 degrees, and the background is relatively clean.
3. My positive control uses positive tissue sections, the negative control is rabitt and rat IgG, and the fluorescent marker control is PBS + fluorescent marker.
4. The serum used in the block is the serum of the animal derived from the secondary antibody, mine is 10% normal donkey serum.
5. The rest is the same as general operation.
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