Immunostaining of Myosin Heavy Chain

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)).