Mouse myod

For cryosections, skeletal muscles were embedded in Tissue-Tek O.C.T. Compound (Gentaur), frozen on 2-methylbutane–cooled liquid nitrogen, and processed for cryostat sectioning. 10-µm sections were collected from the midbelly of muscles. Immunohistochemistry was performed by fixation with 4% PFA/PBS, processing for antigen retrieval with the Antigen Unmasking Solution (Vector) at 95°C for 15 min in a thermostated microwave, permeabilization with 0.2% Triton X-100 in PBS, blocking with 5% heat-inactivated serum/0.2% Triton X-100/1% BSA/PBS, incubation with primary antibody overnight at 4°C, and then incubation with Alexa Fluor secondary antibodies for 1 h at room temperature. Nuclei were counterstained with Hoechst and slides were mounted in fluorescent mounting medium (Dako). Primary antibodies were as follows: mouse β-catenin (BD), goat Collagen Type I (SouthernBiotech), rabbit GFP (Life Technologies), rabbit Ki67 (Abcam), rabbit Laminin (Sigma-Aldrich), mouse MyoD (Dako), mouse embryonic-MyHC (Developmental Studies Hybridoma Bank), mouse Pax7 (DSHB), and rabbit Tcf4 (Cell Signaling Technology). The TUNEL reaction was performed by using the In situ cell death detection kit (Roche) according to the manufacturer’s instructions for tissue cryosections.

Cells were fixed with 4% PFA/PBS, permeabilized with 0.2% Triton X-100/PBS, blocked with 5% heat-inactivated serum/PBS, incubated with primary antibody for at least 1 h, then incubated with Alexa Fluor secondary antibodies for 1 h. Nuclei were counterstained with Hoechst. Primary antibodies are as follows: rabbit APC (Abcam), mouse BrdU (Dako), rabbit activated-Caspase3 (Cell Signaling Technology), rabbit activated β-catenin (Cell Signaling Technology), mouse MyoD (Dako), rabbit Myogenin (Santa Cruz Biotechnology, Inc.), mouse Pax7 (Developmental Studies Hybridoma Bank), and rabbit PCNA (Bethyl Laboratories, Inc.). Secondary antibodies were conjugated with Alexa Fluor 488 or Alexa Fluor 550 fluorochromes. The TUNEL reaction was performed by using the In situ cell death detection kit (Roche) according to the manufacturer’s instructions for adherent cell layers. For cell proliferation studies, cells were incubated for 40 min with culture medium containing 30 µM BrdU and processed for immunostaining with Pax7 and BrdU antibodies after fixation and treatment with 2N HCl for 20 min.