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Immunogold surface replica study on the distribution of acetylcholine receptors in cultured rat myotubes.

We studied the distribution of acetylcholine receptors (AchRs) in rat skeletal muscles, Torpedo membrane fractions, and cultured rat skeletal myotubes. AchRs were first exposed to alpha-bungarotoxin (alpha-BTX) followed by anti-alpha-BTX antibodies. Bound antibodies were visualized with FITC-conjugated goat anti-rabbit antibodies for light microscopy or with peroxidase-conjugated goat anti-rabbit or gold-labeled protein A for electron microscopy. The protocol developed for the present studies detected AchRs with high specificity. In addition, we combined post-fixation immunogold cytochemical and surface replication techniques to study the distribution of AchRs on the dorsal surface of cultured myotubes in detail. Two distinct distribution patterns of AchRs on the cell surfaces of the myotubes were revealed; AchRs were either diffusely distributed or clustered. Dispersed AchRs usually surrounded clusters of AchRs. The AchRs in the clusters were characteristically arranged, and small aggregates of AchRs could also be observed within the clusters. The techniques used in this study are appropriate for studying the dynamics of AchR clustering.