Alexa fluor 488 conjugated anti rabbit igg h l

Cells were washed twice with cold PBS, fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100 in PBS and fixed in blocking solution (6 ng/ml IgG from goat serum in PBS) for 30 min. Cells were incubated with anti-MyHC antibody (1:300, Santa Cruz Biotechnology) overnight at 4 °C. Alexa Fluor 488-conjugated anti-rabbit IgG (H + L) was used as secondary antibody (1:500, Thermo Fisher Scientific). After counterstaining with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI; 500 ng/ml), slides were mounted with Prolong Gold Antifade Reagent (Invitrogen).
Myogenic differentiation was evaluated by calculating differentiation and fusion indexes representing the proportion of nuclei that were localized within MyHC-positive myotubes and the formation of multinucleated myotubes, respectively. Fluorescent images of random fields were captured with 20× magnification. For each experimental condition, total number of nuclei and number of nuclei incorporated into the myotubes were counted. The differentiation index was calculated as the percentage of MHCpositive cells above total and fusion index was determined by the percentage of nuclei incorporated into myotubes (defined as containing at least two nuclei) relative to the total number of nuclei.