A simple immunofluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle--dependent changes in mast cell--nerve fiber contacts in murine skin.
Close contacts between mast cells (MC) and nerve fibers have previously been demonstrated in 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 purpose, we developed the following triple-staining technique. After paraformaldehyde-picric acid perfusion fixation, cryostat sections of back skin from C57BL/6 mice were incubated with a primary rat monoclonal antibody to substance P (SP), followed by incubation with a secondary goat-anti-rat TRITC-conjugated IgG. A rabbit antiserum to CGRP was then applied, followed by a secondary goat-anti-rabbit FITC-conjugated IgG. MCs 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, novel covisualization method, we were able to show that MC-nerve associations in mouse skin are, contrary to previous suggestions, highly selective for nerve fiber types, and that these interactions are regulated in a hair cycle-dependent manner: in telogen and early anagen skin, MCs preferentially contacted CGRP-immunoreactive (IR) or SP/CGRP-IR double-labelled nerve fibers. Compared with telogen values, there was a significant increase in the number of close contacts between MCs and tyrosine hydroxylase-IR fibers during late anagen, and between MCs and peptide histidine-methionine-IR and choline acetyl transferase-IR fibers during catagen.
Botchkarev
V A
VA
Department of Dermatology, Charité, Humboldt-Universität, Berlin, Germany.
Eichmüller
S
S
Peters
E M
EM
Pietsch
P
P
Johansson
O
O
Maurer
M
M
Paus
R
R
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D001360:Avidin / Q000032:analysis; D015740:Calcitonin Gene-Related Peptide / Q000032:analysis; D002450:Cell Communication; D003374:Coumarins / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes; D006197:Hair / Q000254:growth & development*; D006801:Humans; D007150:Immunohistochemistry; D008407:Mast Cells / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009412:Nerve Fibers / Q000502:physiology*; D012235:Rhodamines; D012867:Skin / Q000166:cytology* / Q000294:innervation*; D013373:Substance P / Q000032:analysis
D000911:Antibodies, Monoclonal; D003374:Coumarins; D005456:Fluorescent Dyes; D012235:Rhodamines; D001360:Avidin; D013373:Substance P; C009434:tetramethylrhodamine isothiocyanate; D015740:Calcitonin Gene-Related Peptide; C051029:7-amino-4-methylcoumarin-3-acetic acid