This experimental method was obtained from the official website of the Fourth Military Medical University

Operation method

Immunofluorescence complement assay

Principle

A mixture of specific antibody and complement reacts with the antigen on the specimen, and the complement binds to the antigen-antibody complex, and then the fluorescent anti-complement antibody binds to the complement, thus forming an antigen-antibody-complement complex-anti-complement fluorescent antibody complex, and the site that exhibits a positive fluorescence under the fluorescence microscope is the site where the immune complex exists, and the method is commonly used in diagnosis of renal puncture biopsy and other diagnostic procedures.

Materials and Instruments

Anti-Complement Fluorescent Antibody
PBS Glycerol

Move

1. Specimen processing is the same as that of the direct method → drop appropriately diluted antibody and complement in equal amounts onto the section, put the specimen into a wet box at 37℃ for 30min → wash with PBS twice, 5min each time, stir or vibrate, blow dry → drop appropriately diluted anti-complement fluorescent antibody, stain at 37℃ for 30min → wash with PBS twice, 5min each time, vibrate, wash with distilled water 1min→glycerol buffer sealing solution, fluorescence microscope examination.

2. Control staining

Antigen control, antiserum control and inactivated complement control can be used. After inactivation of complement at 56℃ for 30min, mix the same dilution of complement with equal amount of antibody, and then carry out negative staining by complement method.

Common Problems

Immunofluorescence is now mostly used for clinical tests:

1. Detection of immunoglobulins or immune complexes in tissues, such as kidney puncture specimens in nephritis, skin tissues in lupus erythematosus, etc;

2. to check autoantibodies in blood, in this case, it is often used to use the tissue specimen of mice as the antigen, and after dropping and adding the patient's serum, use the anti-human immunoglobulin antibody with fluorescein to do indirect immunofluorescence method;

3. Identification of cell surface antigens, applicable to tumor cells, lymphocytes, etc., which can be suspended in a tube, labeled and stained, and then transferred to a slide for observation, or classified and quantitatively counted by flow cytometry (FCM);

4. fluorescence tracer study.

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