Goat anti igf 1

Total muscle protein was extracted from the muscle tissue (10 mg) with a solution containing 75 mM Tris-HCl, pH 6.8, 15% SDS, 20% glycerol, 5% dithiothreitol and 0.001% Bromophenol Blue, followed by sonication for 30 s, boiling at 95°C for 5 min and centrifugation at 15,000 g for 5 min. The supernatant was then used for immunoblotting of rapsyn [mouse anti-rapsyn (1:500), Abcam, ab11423], calpain [rabbit anti-calpain II, large subunit (1:500), Merck Millipore, USA, AB81023], BDNF [rabbit anti-BDNF (1:500), Santa Cruz Biotechnology, USA, sc-546], IGF-1 [goat anti-IGF-1 (1:500), Santa Cruz Biotechnology, sc-7144], GDNF [mouse anti-GDNF (1:200), Santa Cruz Biotechnology, sc-13147] and VEGF [rabbit anti-VEGF (1:500), Santa Cruz Biotechnology, sc-507] proteins. For the loading control, protein extracts were run on 6% SDS-PAGE followed by Coomassie Brilliant Blue staining.

Cryosections (8 µM) of EDL muscles were equilibrated with 0.1 M PBS (pH 7.4) at room temperature and post-fixed with 2% PFA for 15 min, followed by blocking in 3% BSA for 2 h. Subsequently, the sections were incubated with the first primary antibody [rabbit anti-BDNF (1:500), Santa Cruz Biotechnology, sc-546 or rabbit anti-GDNF (1:500), Santa Cruz Biotechnology, sc-328] for 48 h. The sections were washed in PBS and incubated with fluorochrome-conjugated secondary antibody [anti-rabbit fluorescein isothiocyanate (FITC) (1:1000) or anti-rabbit Cy3 (1:1000)] for 4 h at room temperature. The sections were re-equilibrated with PBS followed by blocking in 3% BSA for 2 h at room temperature. Further, the sections were incubated with the second primary antibody [goat anti-IGF-1 (1:500), Santa Cruz Biotechnology, sc-7144 or rabbit anti-VEGF (1:500), Santa Cruz Biotechnology, sc-507] for 48 h, washed in PBS and incubated with fluorochrome-conjugated secondary antibody [anti-goat Cy3 (1:1000) or anti-rabbit FITC (1:1000)] at room temperature for 4 h. Stained sections were mounted onto cover slips in 65% glycerol and processed for image capturing with a confocal laser microscope (DMIRE-TCS, Leica, Germany) (Vijayalakshmi et al., 2015 (link)).