Ab156866

50 mg of muscle tissues were homogenized with 1 ml of lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid (EDTA), 1% Triton X-100, 1 mM NaF, 1 mM Na₃VO₄, and 1 mM phenylmethylsulfornyl fluoride (PMSF). After incubation on ice for 30 min, samples were centrifuged at 15,000 rpm for 10 min and subsequently for 30 min at 4 °C. After adjusting the protein concentrations, the supernatants were mixed and boiled with sample buffer containing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol. Samples were electrophoresed with SDS–polyacrylamide gel, transferred to PVDF (polyvinylidene difluoride) membranes and subjected to immunoblotting using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA). The antibodies used were anti-TFG (1:10,000, ab156866, Abcam) and anti-actin (1:2000, sc-47778, Santa Cruz).

The mice were anesthetized, perfused with phosphate buffered saline (PBS) and subsequently with 4% formaldehyde in PBS. Tissues were excised and immediately fixed with 4% formaldehyde in PBS overnight and embedded in paraffin. Sections were cut, deparaffinized, permeabilized with 0.1% Triton and heated in 10 mM citrate buffer (pH 6.0) using a microwave for antigen retrieval. Diaminobenzidine (DAB) staining was performed using a VECTASTAIN Elite ABC Kit (Vector Labs, Burlingame, CA, USA) and ImmPACT DAB (Vector Labs) according to the manufacturer’s instructions. For fluorescent immunostaining, sections were blocked with 3% goat serum in PBS for 1 h, and then incubated with primary antibodies diluted in Can Get Signal Immunostain Solution (Toyobo, Osaka, Japan) overnight at 4 °C. Sections were then incubated with secondary antibodies conjugated with fluorescent dye for 1 h, and then mounted using a Vector TrueVIEW Autofluorescence Quenching Kit (Vector). The primary antibodies used were anti-myosin (skeletal, slow) (1:400, M8421, SIGMA), anti-ChAT (1:5000 for DAB staining, provided by Dr. Hidemi Misawa^{22 (link)}), anti-ChAT (1:1000 for fluorescent immunostaining, MA5-31383, Invitrogen), anti-TFG (1:100, ab156866, Abcam) and anti-OPN (1:100, sc-21742, Santa Cruz).