Immunofluorescence staining

C2C12 cells were transfected in suspension and seeded at high density. At confluency, differentiation was induced using DMEM containing 2% HS in the absence or presence of 5 nM BMP2. After 3 days, cells were fixed with 4% paraformaldehyde, quenched in 50 mM ammonium chloride, permeabilised with 0.2% Triton X-100 in 1x PBS and blocked in 2% BSA/1% FCS. Cells were stained using α-myosin heavy chain (Sigma-Aldrich) and Alexa Fluor^® 488 goat-α-mouse IgG (#A11001, Invitrogen) antibodies. For the staining of differentiated primary foetal myoblasts or satellite cells, α-MF20 (DSHB, #AB_2147781) and Alexa Fluor^® 488 goat-α-mouse IgG or Phalloidin594 Conjugate (sc-363795, Santa Cruz) were used. Nuclei were stained using DAPI (Sigma-Aldrich). Images were acquired by epifluorescence microscopy (Zeiss Axiovert 200), processed using AxioVision software and quantified using ImageJ by creating a threshold mask for the MHC channel with subsequent size gating; the MHC positive area was compared to the sum image area.

Primary myoblasts derived from E18.5 foetuses or primary postnatal satellite cells were seeded on glass cover slips, fixed with 4% PFA and blocked with TSA puffer containing 1x PBS, 10% HS, 0.5% (w/v) blocking reagent (Perkin Elmer) and 0.1% Triton X-100. Cells were stained using an α-IRS4 antibody (Bioss), α-MyoD antibody (BD Biosciences; #554130) or α-Pax7 antibody (DSHB, #AB_528428) and Alexa Fluor 568 goat-α- rabbit or Alexa Fluor 488 donkey –α-mouse antibodies (#A11011; A21202, Invitrogen). Nuclei were stained with DAPI. Images were acquired by confocal microscopy (Zeiss LSM700) and processed using AxioVision software.