Immunofluorescent labeling is a commonly used immunological experimental method in microbiology, immunology, pathology and immunohistochemistry.

Operation method

Immunofluorescence labeling assay of streptavidin-biotin conjugates

Principle

The immunofluorescence technique is a technique in which a known antibody or antigen molecule is labeled with fluorescein, and when it reacts with its corresponding antigen or antibody, a certain amount of fluorescein is carried on the complex formed, and the fluorescent antigen-antibody binding site can be seen under a fluorescence microscope to detect the antigen or antibody. This protocol describes a three-step reaction technique that improves the sensitivity of the immunohistochemical reaction by using a streptavidin-secondary antibody conjugate, which is then reacted with a biotin-fluorescent dye conjugate.

Materials and Instruments

Tissue Samples
Biotin, Fluorescent Dyes, Streptavidin
Centrifuge Shaker

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 the humidified box (the slides should not be in contact with 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 biotinylated secondary antibody was centrifuged at 13,500 g for 2 min at 4°C in a microcentrifuge (40~50 ul antibody per slide).

7. Add the secondary antibody to the slides, place in a humidified box and incubate for 1 h at room temperature.8. Wash slides 3 times with PBS (15 min each wash) and add fluorescent dye-streptavidin to the sections (40-50 ul antibody per slide). Place the slides in a humidified box and incubate at room temperature for 1 h. Wash the slides three times with PBS. 9.

9. Place the coverslip on a paper towel and add 1 drop of Gelvatol to the center of the coverslip. Turn over the slide onto 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.10. 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: PBS + fluorescent marker.

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