Techniques of immunofluorescence and their significance.

1) Skin or mucosal biopsy specimens are usually obtained by 3-5 mm punch or surgical biopsy. 2) Biopsy specimens are snap frozen. If there is a delay between biopsy and snap freezing, the specimen should be kept in cold saline. 3) Frozen 4-6μ sections are cut on a cryostat and placed on a glass slide before being air-dried for 15 minutes. 4) After rinsing in phosphate buffered saline (PBS), pH 7.2, slides are overlaid in a moist chamber with FITC conjugates with the following specificities: antiIgG, antiIgM, antiIgA, antiC3, and antifibrin. Each reagent is tested on a separate slide. 5) After rinsing in PBS, slides are mounted in buffered glycerin and examined under a fluorescence microscope.