Goat anti mouse igg2b alexa555 secondary antibody

Cells were washed 2 × 5 min in phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde/PBS for 15 min. Cells were then washed in PBS (3 × 5 min), permeated with 0.1% TritonX-100/PBS, washed in PBS (3 × 5 min) and then incubated in 3% bovine serum albumin (BSA)/PBS for 2 h. Cells were incubated with primary antibody overnight at 4°C. MF20 (1:50; Developmental Studies Hybridoma Bank, University of Iowa, Department of Biology, Iowa City, IA, USA) in 3% BSA/PBS was used to stain myosin heavy chain (MHC). Cells were then washed with PBS (3 × 5 min) and incubated in goat-anti-mouse IgG2b Alexa555 secondary antibody (1:500, Life Technologies) and DAPI (1:1,000) for 2 h in 3% BSA/PBS. Cells were washed in PBS (3 × 5 min) and then imaged on a Zeiss Axiovert 40 CFL inverted microscope using a 10X objective. Four images were taken in each well from pre-defined locations within each quadrant. Myotube diameter was measured using AxioVision software (AxioVision AC Rel. 4.8.2, Carl Zeiss Imaging Solutions, Wrek, Göttingen; Germany). A total of ~50–80 myotubes were measured per well and the average diameter of each well was used for statistical analysis as described previously (6 (link), 9 (link), 10 (link)).

Cells were rapidly washed three times with ice-cold PBS and fixed at room temperature for 15 min with 4% paraformaldehyde in PBS. The cells were washed three times with PBS and permeated with 0.1% triton X-100 in PBS at room temperature for 10 min. The cells were washed again three times with PBS and blocked with 3% (w/v) of bovine serum albumin in PBS for 2h at room temperature. Cells were incubated with primary antibody overnight at 4°C. MF20 (1:50; Developmental Studies Hybridoma Bank, University of Iowa, Department of Biology, Iowa City, IA, USA) in 3% BSA/PBS was used to stain myosin heavy chain. Cells were then washed with PBS three times for 5 min each, and incubated for 2 h in the dark, in goat-anti-mouse IgG2b Alexa555 secondary antibody (1:1000, Life Technologies) and DAPI (1:1000) in 3% BSA/PBS. Cells were washed in PBS three times for 5 min each and then imaged on a Zeiss Axiovert 40 CFL inverted microscope using a 10X objective. Four images were taken in each well from pre-defined locations within each quadrant (Caldow et al., 2019 (link)).