Ab125078

RH30 cells or myoblasts were fixed in 4% formaldehyde (POCH) in PBS, permeabilized in 0.1% TritonX-100 (Sigma-Aldrich), blocked in 1% bovine serum albumin (BSA, Sigma-Aldrich), incubated with rabbit anti-MYF5 monoclonal antibody (ab125078, Abcam) or anti-fast myosin skeletal heavy chain antibody (MyHC, MY-32, ab51263, Abcam), and then incubated with secondary goat anti-rabbit or anti-mouse antibodies conjugated with Alexa Fluor 555 (Life Technologies) and sometimes with Hoechst. The stained slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories Inc. Burlingame, CA, USA) or Dako Fluorescence Mounting Medium (Dako, Denmark). For visualization of morphology, the cells were stained with Wright’s dye (Sigma-Aldrich). Labeling was assessed by fluorescence microscopy using an Olympus BX51 or IX70 microscope (Olympus Corporation, Tokyo, Japan) and Olympus XC50 camera with cellSens Dimension software (both from Olympus). The images were processed using cellSens Dimension software or ImageJ software (National Institute of Health, USA). To quantify RH30 cells and myoblasts fusion, we calculated the fusion index by expressing the number of nuclei within MyHC-positive cells with ≥2 nuclei as a percentage of the total nuclei.

Primary antibodies: Myh2 (3 μg/ml, Developmental Studies Hybridoma Bank, sc-71, AB_2147165), Myh4 (3 μg/ml, Developmental Studies Hybridoma Bank, BF-F3, AB_2266724), Myh7 (3 μg/ml, Developmental Studies Hybridoma Bank, BA-F8, AB_10572253), Desmin (1:20. Thermo Fisher Scientific, MA5-13259), Cox5a (1:1000, Abcam, ab110262, AB_10861723), Glut1 (1:200, Abcam, ab652, AB_305540), Myf5 (1:10,000, Abcam, ab125078, AB_10975611), Fasn (1:1000, Abcam, ab22759, AB_732316). Secondary antibodies: goat-anti-mouse IgG2b Alexa 350 (1:200, Thermo Fisher A-21140, AB_2535777), goat-anti-mouse IgG1 Alexa 488 (1:200, Thermo Fisher A-21121, AB_2535764), goat-anti-mouse IgM Alexa 555 (1:200, Thermo Fisher A-21426, AB_2535847), IRDye 800CW goat anti-mouse IgG Secondary Antibody (1:10,000, Licor, 926-32210, AB_621842), IRDye 800CW goat anti-rabbit IgG Secondary Antibody (1:10,000, Licor, 926-32211, AB_621843), Anti-rabbit IgG HRP-linked Antibody (1:200, Cell Signaling Technology, 7074P, AB_2099233).