Muscle creatine kinase

Cell or myobundle protein was isolated in RIPA lysis and extraction buffer with protease inhibitor (Sigma). Protein concentration was determined using BCA assay (Pierce of Thermo Scientific, Rockford, IL) according to manufacturer's instructions. Western blot was performed using Bio-Rad Mini-PROTEAN gels and the Mini-PROTEAN Tetra cell, Mini Trans-blot module (Bio-Rad, Hercules, CA). The following primary antibodies were used for detection: GAPDH (SCBT, 1:500), LC3 (Cell Signaling, 1:200), muscle creatine kinase (SCBT, 1:200), myosin heavy chain 1/2/4/6 (SCBT, 1:200), and sarcomeric alpha-actinin (Sigma, 1:200). HRP conjugated anti-mouse (1:20,000) and anti-goat (1:5000) antibody were purchased from Sigma, and HRP conjugated anti-rabbit was purchased from SCBT (1:5000). Chemiluminescence was performed using Clarity Western ECL substrate (Bio-Rad). Images were acquired using a Bio-Rad Chemidoc and analyzed using ImageJ.

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.