Direct immunofluorescence enables detecting antibodies, antigens, complement components and fibrin. Antibodies marked by a fluorochrome (eg. fluoresceinisothiocyanate, FITC) — conjugates — react with the tissue. After washing unbound antibodies out the tissue is evaluated using the fluorescent microscope. Labeled antibody-antigen complexes shine yellow-green. The contrast can further increase the staining by the Evans red (the background is reddish).

The tissue for evaluation is obtained by the 4 mm punch biopsy. Fast freezing of the tissue is extremely important. If immediate freezing is not possible, special transporting medium (maleinimide) can be used.

Direct immunofluorescence is rarely diagnostic (eg. IgG and c3 intercellulary together with the acantholytic bula in pemphigus). In most cases the combination of different immunoglobulines and complement factors together with results of classic transmitted light histologic evaluation enables specific diagnosis.

The site of biopsy is very important. In bullous or vesicular diseases its is the border of a vesicle, in lupus erythematodes and porphyria the sun exposed areas where the changes are suitable for diagnosis. In excisions of systemic disorders on the other hand the biopsy should be made from sun protected areas (volar areas of the forearm, buttocks) because of possibility of false positive results.

Uninvolved (“normal”) skin examination is important especially in systemic lupus erythematodes. Examination done to early can be negative. Biopsy should always follow detailed clinical and laboratory examination.