Ab2868

Cryosections (8–10-mm thickness) were prepared from frozen skeletal and cardiac muscles and formalin-fixed paraffin-embedded liver. Histological staining was performed with hematoxylin-phloxine-saffron.
Immunodetection was performed with the monoclonal antibody dysferlin-Hamlet (NCL-Hamlet, diluted 1:50) as described previously^{28 (link)} or with the monoclonal dysferlin-Romeo (ab124684; Abcam) diluted 1:50. Mouse anti-CD11b (550282; BD Biosciences; Le Pont de Claix, France); diluted 1:40 was used to detect inflammatory cells and RyR (ab2868; abcam) diluted 1:200 was used to perform colocalization of dysferlin in T-tubules. Sections were mounted with DAPI-Fluoromount-G (Southern Biotech, Birmingham, AL) and examined under a confocal microscope Leica TCS SP2.
Evans Blue dye positive fibers were revealed by fluorescence excitation at 633 nm on a Leica confocal fluorescent microscope on cryosections treated 10 minutes in frozen acetone. Sections were mounted with Fluoromount-G (Southern Biotech) before the observation. The cartography of the whole section was performed using a motorized stage at an original 40x magnification and using the software Cartograph (Microvision, Evry, France). The ratio of the area corresponding to the Evans Blue positive cells versus the whole area of the section was measured using Image J in the red channel.

Tissue was homogenized in homogenization buffer containing (in mm): HEPES 20, NaCl 150, EDTA 5, KF 25, sodium orthovanadate 1 with glycerol 20%, triton X‐100 0.5% and protease inhibitor cocktail (Roche, cOmplete), pH 6.8. Anti‐RyR antibody (Abcam, ab2868) was incubated with Dynabeads Protein G (Invitrogen) for 40 min at room temperature (1 μl antibody + 11 μl beads per sample). The beads were washed with PBS‐0.05% Tween‐20 twice and homogenization buffer once. A 400 μl aliquot of lysate (total protein concentration of 1 mg ml⁻¹) was incubated at 4°C overnight with antibody‐bead in homogenization buffer. After washing, electrophoresis was performed using protein gels of 8%. Proteins were transferred to PVDF membranes using the Trans‐Blot semi‐dry transfer system (BioRad). Membranes were blocked for 1 h in PBS‐0.05% Tween‐20 solution containing 5% dried skimmed milk. Antibodies used were RyR2 (Abcam, ab2868, 1:5000) and pSer2808 RyR2 (Badrilla, A010‐30AP, 1:1000). Data were normalized to RyR in the immunoprecipitated sample.