Anti desmin primary antibody

For whole mount X. laevis experiments, 3-day-old X. laevis tadpoles were fixed in Dent's fixative (80% methanol, 20% dimethyl sulfoxide) at −20 °C overnight, followed by bleaching and labelling, as previously described [44 (link)]. The anti-desmin primary antibody (1:13, Sigma-Aldrich) was followed by anti-mouse peroxidase-conjugated IgG (1:3500, Dianova), and antibody interaction visualization using diaminobenzidine.

C2C12 myotubes were fixed in PBS containing 4% paraformaldehyde and permeabilized with PBS 0.5% triton X‐100. The preparation was incubated with an anti‐desmin primary antibody (1/100; Sigma‐Aldrich) diluted in PBS/BSA for 1 h at 37°C then washed in PBS, followed by a 30 min incubation with fluorescein‐conjugated anti‐mouse (1/100; Interchim Fluoprobes 488). Nuclei were stained with Hoechst (0.1 mg/mL; Sigma). The slides were examined with an Axiovert 40 fluorescent microscope (Carl Zeiss, Oberkochen, Germany). To estimate myotube size, the diameter of at least 500 myotubes per condition in at least three independent cultures was measured using the ZEN lite software (Carl Zeiss, Oberkochen, Germany). The average diameter per myotube was calculated as the mean of three measurements taken along the length of the myotube. The fusion index is defined as the proportion of cells that contain three or more nuclei. The fusion index was determined after 5 days of differentiation by counting at least 1000 nuclei per condition and per culture in three independent cultures.