Alex fluor 488 conjugated alpha bungarotoxin

Enzymatically isolated single muscle fibers were fixed with 4% PFA for 20 minutes at RT. After the fixation 0.1 M glycine in PBS was used to neutralize excess formaldehyde. Fibers were permeabilized with 0.5% Triton-X in PBS (PBST) for 10 minutes, blocked with a serum-free Protein blocking solution (DAKO, Los Altos, CA, USA) for 30 minutes and rinsed three times with PBST solution. Anti-Septin7 (JP18991, IBL, Hamburg, Germany) diluted in blocking solution was added and the fibers incubated overnight at 4 °C in a humid chamber. Samples were washed three times with PBST and incubated with Alex-Fluor 488 conjugated alpha-bungarotoxin (1:500 dilution, B13422, Thermo Fisher) and Cy-3 conjugated anti rabbit IgG at (dilution 1:300 ) (A10520, Thermo Fisher) for 1 hour at room temperature. After washing three times drop slides were mounted with DAPI containing mounting medium (H-1200–10, Vector Laboratories, Burlingame, CA, USA). Images were acquired with a Zeiss AiryScan 880 laser scanning confocal microscope.

Enzymatically isolated single muscle fibers were fixed with 4% PFA for 20 minutes at RT. After the fixation 0.1 M glycine in PBS was used to neutralize excess formaldehyde. Fibers were permeabilized with 0.5% Triton-X in PBS (PBST) for 10 minutes, blocked with a serum-free Protein blocking solution (DAKO, Los Altos, CA, USA) for 30 minutes and rinsed three times with PBST solution. Anti-Septin7 (JP18991, IBL, Hamburg, Germany) diluted in blocking solution was added and the fibers incubated overnight at 4 °C in a humid chamber. Samples were washed three times with PBST and incubated with Alex-Fluor 488 conjugated alpha-bungarotoxin (1:500 dilution, B13422, Thermo Fisher) and Cy-3 conjugated anti rabbit IgG at (dilution 1:300 ) (A10520, Thermo Fisher) for 1 hour at room temperature. After washing three times drop slides were mounted with DAPI containing mounting medium (H-1200-10, Vector Laboratories, Burlingame, CA, USA). Images were acquired with a Zeiss AiryScan 880 laser scanning confocal microscope.