A SIMPLE IMMUNOFLUORESCENCE TECHNIQUE FOR SIMULTANEOUS VISUALIZATION OF MAST-CELLS AND NERVE-FIBERS REVEALS SELECTIVITY AND HAIR CYCLE - DEPENDENT CHANGES IN MAD CELL - NERVE-FIBER CONTACTS IN MURINE SKIN

Close contacts between mast cells (MC) and nerve fibers have previousl y been demonstrated hi normal and inflamed skin by light and electron microscopy, A key step for any study in MC-nerve interactions in situ is to simultaneously visualize both communication partners, preferably with the option of double labelling the nerve fibers, For this purpos e, we developed the following triple-staining technique, After parafor maldehyde-picric acid perfusion fixation, cryostat sections of back sk in from C57BL/6 mice were incubated with a primary rat monoclonal anti body to substance P (SP), followed by incubation with a secondary goat -anti-rat TRITC-conjugated IgG. A rabbit antiserum to CGRP was then ap plied, followed by a secondary goat-anti-rabbit FITC-conjugated IgG, M Cs were visualized by incubation with AMCA-labelled avidin, or (for a more convenient quantification of close MC-nerve fiber contacts) with a mixture of TRITC- and FITC-labelled avidins. Using this simple, nove l co-visualization method, we were able to show that MC-nerve associat ions in mouse skin are, contrary to previous suggestions, highly selec tive for nerve Fiber types, and that these interactions are regulated in a hair cycle-dependent manner: in telogen and early anagen skin, MC s preferentially contacted CGRP-immunoreactive (IR) or SP/CGRP-IR doub le-labelled nerve fibers, Compared with telogen values, there was a si gnificant increase in the number of close contacts between MCs and tyr osine hydroxylase-IR fibers during late anagen, and between MCs and pe ptide histidine-methionine-IR and choline acetyl transferase-la fibers during catagen.