Goat anti brn3a polyclonal antibody

Eyes were placed into fixative consisting of 4% paraformaldehyde (PFA) in PBS for 1 hour, at room temperature, immediately after enucleation. The whole retinas were isolated and placed in blocking buffer (PBS containing 2% goat serum, 2% donkey serum, 3% BSA, and 2% Triton) for 1 hour at room temperature, and subsequently incubated with goat anti-Brn3a polyclonal antibody (Santa Cruz Biotechnology,sc-31984, 1:200) in blocking buffer overnight at 4°C on a vacillating platform. Following this, retinas were washed 3 times for 30 min in PBS containing 0.3% Triton and incubated with the secondary antibody Alexa Fluor 488 (Alexa Fluor 488 conjugated donkey anti-goat IgG, 1:500) for 2 hours at room temperature. For Tuj-1 staining, retinas were incubated with rabbit anti-beta Ⅲ Tubulin polyclonal antibody (Abcam:18207, 1:300) in blocking buffer overnight at 4°C, washed 3 times for 30 min in PBS containing 2% Triton, and incubated with Alexa Fluor 488 (Alexa Fluor 488 conjugated donkey anti-rabbit IgG, 1:500) for 2 hours at room temperature. For staining of the retinal vasculature, retinas were incubated with Isolectin GS-IB4 (Alexa Fluor 647, 1:100) in blocking buffer overnight at 4°C. Prior to flat-mounting (described below), the retinas were again washed 3 times for 30 minutes in PBS.