Desmin

The following antibodies were used for immunofluorescence or western blot: pan-MyHC (MF20; DSHB), Titin (9D10; DSHB), MyHC-neo (N3.36; DSHB) (MHCN; Leica), MYH3 (F1.652; DSHB), MYH1/2 (SC-71; DSHB), α-actinin (MA122863; Thermofisher), Desmin (sc-23879; SCBT), ACTB (sc-4778; SCBT), OCT3/4 (C-10; SCBT), SOX2 (Y-17; SCBT), NANOG (H-2; SCBT), Alexa Fluor 488 Phalloidin (A12379; Thermofisher), Alexa fluor 555 goat anti-mouse IgG (A-21424; Thermo Fisher), Alexa fluor 488 goat anti-rabbit IgG (A-11008; Thermo Fisher), and mouse IgG HRP-linked (NA931; GE Healthcare).

Cells were fixed in 4% paraformaldehyde in PBS for 10 min and myobundles were fixed in 2% paraformaldehyde in PBS overnight at 4°C. Following fixation, samples were washed in PBS then blocked in 5% chick serum with 0.2% Triton-X 100. The following primary antibodies were used for tissue characterization: desmin (SCBT, Dallas, TX, 1:200), anti-GFP (Life Technologies, 1:200), laminin (Abcam, Cambridge, MA, 1:200), muscle creatine kinase (SCBT, 1:100), MyoD (BD, 1:100), myogenin (SCBT, 1:100), myosin heavy chain 1/2/4/6 (SCBT, 1:100), Pax7 (DSHB, Iowa City, IA, 1:50), sarcomeric α-actinin (Sigma, 1:200), and vimentin (Sigma, 1:200). Corresponding fluorescently labeled secondary antibodies (1:200), α-bungarotoxin (1:100), and phalloidin (1:200) were purchased from Life Technologies. Oil Red O staining was performed using standard protocols on cryosections of myobundles fixed in 4% paraformaldehyde. Hematoxylin and eosin stain was performed on paraffin embedded sections of 2% paraformaldehyde fixed myobundles using Harris modified hematoxylin (Sigma) and Eosin Y (Sigma). Images were acquired using a Zeiss 510 inverted confocal microscope and analyzed using LSM Image Software. Mosaic images for fiber length measurements were generated using Mosaic J in FIJI.