Extensor digitorum longus (EDL) muscles were dissected, pinned in mild stretch, and fixed by immersion for 20 minutes in 4% paraformaldehyde with PBS (pH 7.4). After rinsing in PBS, muscles were soaked in 15% sucrose with PBS (overnight at 4°C), followed by 30% sucrose with PBS for at least 24 hours. Muscle tissues were embedded in OCT and sectioned with a cryostat (HM 550; Microm GmbH) into 40-mm sections and placed on glass slides for staining. For a presynaptic marker, sections were incubated with βIII-tubulin (1:500; Promega, catalog G7121) antibody overnight at 4°C, followed by secondary fluorescein isothiocyanate–labeled goat anti-mouse (1:100; Jackson ImmunoResearch, catalog 115-095-205) antibody. Rhodamine bungarotoxin (1:40, Thermo Fisher Scientific,, catalog B13423) was used as a postsynaptic marker. Stained sections were examined under fluorescence and confocal microscopy. The Zeiss LSM Image Examiner version 1.0.0.241 (Carl Zeiss) was used for imaging analysis. We quantitated pre- over postsynaptic NMJ areas, and this method has shown to be effective in detecting differences in NMJ connectivity in published studies (64 (link)).
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