Ab8592

Materials and analytical-grade chemicals used for the mass spectrometric analysis of WR versus WT (wild-type) skeletal muscle tissues were obtained from Amersham Biosciences/GE Healthcare and BioRad Laboratories. The proteolytic enzymes Lys-C and trypsin were purchased from Promega. Protease inhibitors and chemiluminescence substrate were from Roche Diagnostics. Whatman nitrocellulose transfer membranes were obtained from Invitrogen. Primary antibodies were purchased from Abcam [ab55559 to MBP (myosin-binding protein) C; ab97708 to troponin TnT; ab48003 to MLC2 (myosin light-chain 2); ab8592 to desmin; ab41803 to annexin; ab26300 to lamin; ab6588 to collagen; ab13496 to αB-crystallin; ab85366 to carbonic anhydrase CA3; ab43176 to ATP-synthase; ab36329 to isocitrate dehydrogenase; and ab28172 to prohibitin] and Sigma Chemical Company (L-9393 to laminin). Secondary peroxidase-conjugated secondary antibodies were obtained from Chemicon International. All other chemicals were analytical grade and purchased from Sigma Chemical Company.

Dissection and whole mount staining of retinas were performed as previously described [28 (link)]. Enucleated eyes were fixed for 1 h in 4% paraformaldehyde, rinsed three times in PBS, dissected and stored in methanol at −20°C. Immunohistochemistry of whole-mount samples was performed by using isolectin IB4 (Iso B4; Thermo Fisher Scientific, catalog #I21411, 1:200), rabbit anti-desmin antibody (Abcam, catalog #ab8592, 1:100), mouse anti- α-SMA (Sigma, catalog #F3777, 1:100), rabbit anti-collagen IV (Abcam, catalog #ab6586, 1:100) and rabbit anti-CD31 (Abcam, catalog # ab28364, 1:100). For detection, suitable specific Alexa Fluor-coupled secondary antibodies were used (Thermo Fisher Scientific, 1:1000). For the in vivo BrdU incorporation assay, 100 μg of BrdU (BD Pharmingen) per gram of body weight was injected intraperitoneally 4 h before the mice were euthanized with CO₂ followed by cervical dislocation. Retinas were isolated and collected for analysis as above. BrdU positive cells were stained by mouse anti-BrdU antibody (Cell signaling, catalog #5292, 1:100).