Plastic-embedded semithin cross-sections as a tool for high-resolution immunofluorescence analysis of the neuromuscular junction molecules: Specific cellular location of protease-activated receptor-1.
In the neuromuscular junction (NMJ), three cellular elements (nerve ending, postsynaptic muscle component, and teloglial Schwann cell) are closely juxtaposed and functionally interdependent. It is important to determine the precise location of the relevant molecules involved in structural stability and neurotransmission at the three cellular components of this synapse in order to understand the molecular mechanisms underlying NMJ formation, maintenance, and functionality. In this paper, we show that plastic-embedded 0.5-mum semithin cross-sections from whole-mount multiple-immunofluorescence-stained muscles provide a simple and sensitive high-resolution procedure for analyzing the cellular and subcellular distribution of molecules at the NMJ. We have used this procedure to resolve the location of protease-activated receptor 1 (PAR-1). Previously, by immunohistochemistry we had detected PAR-1 in muscle fibers concentrated in the synaptic area but could not determine whether PAR-1 is expressed only in the muscle fiber at the NMJ. Our present results demonstrate that PAR-1 is concentrated in the postsynaptic region but not in the presynaptic terminal and that the labelling pattern for PAR-1 overlapped with Schwann cell staining.
Lanuza
Maria A
MA
Unitat d'Histologia i Neurobiologia (UHN), Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain. mariaangel.lanuza@urv.net
Besalduch
Núria
N
Garcia
Neus
N
Sabaté
Mar
M
Santafé
Manel M
MM
Tomàs
Josep
J
D000818:Animals; D005455:Fluorescent Antibody Technique / Q000379:methods*; D066298:In Vitro Techniques; D046529:Microscopy, Electron, Transmission / Q000379:methods; D018485:Muscle Fibers, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009469:Neuromuscular Junction / Q000378:metabolism* / Q000648:ultrastructure; D016613:Plastic Embedding / Q000379:methods*; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D044463:Receptor, PAR-1 / Q000378:metabolism*; D012583:Schwann Cells / Q000378:metabolism / Q000648:ultrastructure
D009419:Nerve Tissue Proteins; D044463:Receptor, PAR-1