Methodological aspects on immunohistochemistry in dermatology with special reference to neuronal markers

In an attempt to optimize the immunohistochemical procedure for visualizing neuronal markers, such as neuropeptides, in the human skin, different alternatives in all steps of the process were compared. We have studied the influence of type of immunohistochemical method, the biopsy technique, including the size of the punch biopsy, anaesthesia, the choice of fixative and the time of fixation, the storage process, the sectioning parameters, incubation procedure, the type of fluorophore-conjugated antibody and its dilution, mounting and storage, and, finally, microscopical examination. The following procedure was found to give the best result: punch biopsies of 3 mm, taken under local anaesthesia using lidocaine injected into the dermis-subcutis at the place of biopsy; fixation by a buffered 10% formalin solution containing 14% of saturated picric acid for 2 h at 4 degrees C; storage in 10% sucrose buffer for at least 24 h up to 1 month at 4 degrees C or deep-frozen to -70 degrees C for 2 months (with only a minor structural deterioration); cryostat sectioning of the biopsies with a section thickness of 14 microns and with the cutting edge perpendicular to the skin surface; rhodamine (TRITC)-conjugated, instead of fluorescein-isothiocyanate (FITC)-conjugated, secondary antiserum, since it gives a lower background fluorescence; and for the incubation and mounting procedures, our standard laboratory routines were applied. The result is an optimal indirect immunofluorescence technique, to be applied in dermatology. We also found that biopsies taken under local anaesthesia with chloroethyl spray lost almost all immunofluorescence for several neuronal markers in the epidermis-upper dermis.