Immunofluorescence labeling of tissue sections

Summary

The immunofluorescence technique is the labeling of a known antibody or antigen molecule with fluorescein, which can be used for (1) examination of antigen-antibody binding sites (2) immunological and histological testing.

Operation method

immunofluorescent marker

Principle

Immunofluorescence technique is to label a known antibody or antigen molecule with fluorescein, and when it reacts with its corresponding antigen or antibody, a certain amount of fluorescein will be carried on the complex formed, and the antigen-antibody binding site emitting fluorescence can be seen under the fluorescence microscope, so that the antigen or antibody can be detected.

Materials and Instruments

Tissue samples
PBS Antibody
slides humidifying cassettes

Move

1. Wet paper towels are placed on the bottom of the slide box to make a humidified box, and the slides containing the sections are taken out of the cryostat and placed into the slide box (6 slides on each side) or into the humidified box (the slides should not touch each other).2. When the slides reach room humidity and are not dry, spread PBS on the slides (do not overflow the slides).3. Centrifuge the diluted first antibody in a microcentrifuge at 13,500 g for 2 min at 4°C (40-50 ul of antibody should be added to each slide to cover the slides).4. Using a Pasteur pipette attached to the pump, aspirate the PBS from the slide at one end of the section and add the antibody from the other end, cover with a humidifying cassette, and incubate for 1 h at room temperature.
5. PBS the slide 3 times (5 min/time), add new PBS buffer from one end of the section, and aspirate the old buffer from the other end.6. Diluted secondary antibody was centrifuged at 13,500 g in a microcentrifuge at 4°C for 2 min (40~50 ul antibody per slide).7. The secondary antibody was added to the sections and incubated in a humidified box at room temperature for 1 h. The slides were washed with PBS 3 times (5 min/time).

Figure I. Various methods of immunohistochemical labeling
8. Place the coverslip on a paper towel and add 1 drop of Gelvatol to the center of the coverslip. Turn over the slide and place it on the coverslip (without pressure), place the slide on a bench, cover with aluminum foil and leave it in the light for 30 min to allow the Gelvatol to condense.
9. Observe the results under a microscope or store in a slide box at 4°C.

Caveat

1、Fluorescence staining is usually observed within 1h, or stored at 4℃ for 4h, too long a time may cause fluorescence to decline prematurely.

2、The following three controls should be set for each test：

（1） Positive control: positive serum + fluorescent marker; (2) Negative control: positive serum + fluorescent marker.(2) Negative control: negative serum + fluorescent marker; (3) Fluorescent marker control: negative serum + fluorescent marker.(3) Fluorescent marker control: PBS + fluorescent marker.

Common Problems

The basic procedure for specimen preparation in immunofluorescence is similar to that of enzyme immunohistochemistry, with the following differences:

1, immunofluorescence does not require the use of hydrogen peroxide treatment, closure and primary antibody incubation and the same.

2. The secondary antibody for immunofluorescence is incubated with different fluorescent labeled secondary antibodies, and the incubation time is determined according to the working concentration of the antibody.

3、After the secondary antibody incubation, the film can be washed sufficiently, and then it can be patched, sealed and observed.

4, immunofluorescence in the sealing of the film is often used special sealer or glycerol: 0.01M PBS (1:1). If conditions permit, it is recommended to purchase quench-resistant sealing solution, so that the specimen can be stored for a longer period of time.

5. Incubation of fluorescent antibody and subsequent processing should be protected from light.

6. False positives of fluorescent antibody staining may be numerous, and positive and negative controls need to be set separately.

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