Fluoromount medium fluromount g

Co-cultures were washed twice with PBS (Gibco ref 14190-094). Fixation was done in either 4% paraformaldehyde (Electron Microscopy Science, 15710-S) for 20 min at room temperature (RT) or in acetone/methanol solution (ratio 1:1) for 6 min at −20°C according to the specific requirements of each antibody. Alpha-bungarotoxin (BTX) labeling of AChRs was performed with 5 µg/ml TRITC-BTX (Sigma-Aldrich, T0195) in PBS for 15 min at RT prior to permeabilization and prior to acetone/methanol fixation. After BTX staining, cells were washed twice with PBS. Permeabilization was performed in PBS 5% Triton X-100 (Sigma-Aldrich, X100) for 5 min at RT. Cells were washed twice in PBS and then saturated in PBS containing 5% bovine serum albumin (BSA) and 10% goat serum (Gibco, 16210-064) for 1 h at RT. Primary antibodies were incubated overnight at 4°C in PBS containing 5% BSA and 0.1% saponin. Co-cultures were washed 3×10 min with PBS at RT under slow agitation and stained with the corresponding secondary antibodies supplemented with DAPI for 1 h at RT. Co-cultures were washed 3×10 min with PBS at RT and then mounted in Fluoromount medium (Fluromount-G, Southern Biotech, 0100-01) and analyzed using confocal microscopy (Leica SPE confocal microscope with a 63×1.3 NA Apo objective).

The same protocol for cultured cells or isolated fibers was used. Cells were washed twice with PBS (Gibco ref 14190-094). Fixation was done in either 4% PFA (Electron Microscopy Science ref 15710-S) for 20 minutes at room temperature (RT) or in acetone/methanol solution (ratio 1:1) for 6 minutes at −20 °C accordingly to antibodies’ specific requirements. α-bungarotoxin labeling of AChRs was performed with 5 μg/ml TRITC-BTX (Sigma Aldrich ref T0195) in PBS for 15 minutes at RT prior to permeabilization and prior to acetone/methanol fixation. Following permeabilization in PBS 5%Triton (Sigma Aldrich ref X100) for 5 minutes at RT, cells were washed twice in PBS and then saturated in BSA 5%, 10% goat serum (Gibco ref 16210-064) for 1 hour at RT. Primary antibodies were incubated overnight at 4 °C in PBS BSA 5%, saponin 0.1%. Cells were washed with PBS at RT and stained with corresponding secondary antibodies supplemented with DAPI for 1 hour at RT. Cells were washed with PBS at RT and then mounted in Fluoromount medium (Fluromount-G, Southern Biotech, ref 0100-01) and analyzed.