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

Abstract

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.

Synaptic activity‐related classical protein kinase C isoform localization in the adult rat neuromuscular synapse

“…It stained the nerve terminals in the NMJ as in previous reports (see Figs. 3, 8;Santo Neto et al, 2003;Lanuza et al, 2007 Chao et al, 2008). It stained the nerve terminals in the NMJ as in previous reports (see Figs.…”

“…It stained nerve terminal boutons in the NMJ as in previous reports (see Figs. 3, 8;Prasarnpun et al, 2004;Lanuza et al, 2007). The polyclonal antisyntaxin antibody (Sigma) detected the same bands as the monoclonal antisyntaxin clone HPC-1 antibody (Perez-Legaspi et al, 2008) and stained nerve terminals boutons in the NMJ as in previous reports (see Figs.…”

“…It stained the nerve terminals in the NMJ as in previous reports (see Figs. 3, 8;Lanuza et al, 2007). Rabbit anti-S100 antibody (Dako, Carpinteria, CA) detected a single band in human kidney of 10.5 kDa on Western blot (Lin et al, 2006).…”

“…The polyclonal antisyntaxin antibody (Sigma) detected the same bands as the monoclonal antisyntaxin clone HPC-1 antibody (Perez-Legaspi et al, 2008) and stained nerve terminals boutons in the NMJ as in previous reports (see Figs. 3, 8;Lanuza et al, 2007). The monoclonal antineurofilament-200 antibody (Sigma) detected a single band in monkey retina of 200 kDa (Kashiwagi et al, 2003).…”

“…It stained the Schwann cells in the NMJ as in previous reports (see Figs. 3, 8;Zuo et al, 2004;Lanuza et al, 2007). The mouse anti-S100 antibody (Acris, Hiddenhausen, Germany) detected a single band of 11 kDa (manufacturer's information).…”