Uplsapo20x na 0.75 objective

EDL and soleus muscles were dissected and freed from surrounding tissues, the tendons pinned at the bottom of a plastic histology mold, the muscle cryopreserved in 5% sucrose overnight, followed by 20% sucrose overnight, then washed in PBS and covered with optimal cutting temperature compound (O.C.T.), and frozen at −80 °C until used. In preliminary experiments, we exposed muscles sections to a buffer containing the calcium chelator 3% ethylenediaminetetraacetic acid (EDTA) in PBS to prevent contraction; however, they did not show significant differences in the muscle and NMJ organization compared to those in control conditions devoid of EDTA. Muscles were longitudinally sectioned (25 μm) with a Leica CM3050S cryostat, mounted on glass slides, fixed in 2% PFA for 30 min at room temperature, washed in PBST, then in PBS, incubated in 1:250 in PBS tetramethylrhodamine 554-α-bungarotoxin-conjugate for 3 h, rinsed in PBS, and mounted using Dako mounting medium. The preparation was imaged using an Olympus FV1200 spectral laser scanning confocal microscope, an UPLSAPO20X NA: 0.75 objective, and Olympus MetaMorph software, at 2 μs/pixel, and 0.31 μm/pixel.