Lsm5 pascal laser scanning confocal microscopy

Cells were cultured on round cover slips coated with Matrigel (Corning, New York, NY, USA), diluted with DMEM. Before and after myotube formation, cells were fixed with 4% paraformaldehyde or ice-cold methanol for 10 min. Next, cells were permeabilized and blocked with phosphate-buffered saline (PBS) containing 1 mg/ml BSA and 0.1% Triton X-100 for 15 min at 25 °C. The cells were then incubated with primary antibodies for 1 h at 25 °C or overnight at 4 °C. Cells were washed in PBS with 0.1% Triton X-100 three times, incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA), diluted 1:400 in PBS, counterstained with Hoechst 33342, and mounted. The primary antibodies used were as follows: anti-GGA1 or 2 (this study), anti-Golgin subfamily A member 2 (GM130), anti-Ras-related protein rab-4A (RAB4), anti-clathrin heavy chain (CHC), and anti-adaptor related protein complex 1 subunit gamma 1 (AP1G1) antibodies were from BD Biosciences (San Jose, CA, USA), whereas anti-lysosomal associated membrane protein 1 (LAMP1; 1D4B) rat monoclonal antibody, anti-MYH3, and anti-beta actin antibodies were purchased from Santa Cruz (Dallas, TX, USA). The immunofluorescent images were analyzed by BZ-9000 (Keyence, Osaka, Japan) and LSM5 Pascal laser scanning confocal microscopy (Zeiss, Jena, Germany).