Alexa fluor 488 conjugated chicken anti rabbit

Mice were sacrificed at P6. Eyeballs were enucleated and fixed with 4% PFA at room temperature for 20 min followed by a cold 70% ethanol fixation at −20°C overnight. Retinas were dissected and stained overnight at 4°C with primary antibodies including CD31 antibodies (1:500; R&D Systems; AF3628), MFSD2A antibodies (1:500; a gift from D. Silver at Duke-NUS Graduate Medical School, Singapore), and CAV-1 (Cell Signaling Technology, Danvers, MA; 3267). Retinas were then washed and stained with secondary antibodies including Alexa Fluor 488–conjugated chicken anti-rabbit (Thermo Fisher Scientific; A-21441) and Alexa Fluor 594–conjugated donkey anti-goat (Thermo Fisher Scientific; A-11058) at room temperature for 1 hour, followed by whole mounting and imaging with a confocal microscope (LSM 880, Zeiss).

Differentiated human myotubes grown on a glass coverslip coated with laminin were fixed with 4% paraformaldehyde (made in PBS), permeabilized with 1% Triton in PBS for 20 min, and processed as previously described (Treves et al., 2011 (link)). The following antibodies were used: mouse anti-RyR1 (MA3-925; Thermo Fisher Scientific), goat anti-Ca_v1.1 (sc-8160; Santa Cruz Biotechnology, Inc.), rabbit anti-Ca_v1.2 (sc-25686; Santa Cruz Biotechnology, Inc.), mouse anti-dystrophin (ab-7164; Abcam), rabbit anti-utrophin (sc-15377; Santa Cruz Biotechnology, Inc.), Alexa Fluor 488–conjugated chicken anti–rabbit, Alexa Fluor 555–conjugated donkey anti–goat IgG (Thermo Fisher Scientific), and Alexa Fluor 647–conjugated goat anti–mouse IgG (Thermo Fisher Scientific). Cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) to visualize nuclei and observed using an A1R confocal microscope (Nikon) with a CFI Apo TIRF 100× (1.49 NA) objective.