Goat anti brn3a antibody sc 31984

All reagents were purchased from Sigma (Poole, UK) unless otherwise specified. Frozen tissue sections were left for 30 min to equilibrate to room temperature and then hydrated in PBS 3 × 5 min before being immersed in 0.1% Triton X-100 for 20 min to permeabilise the sections. Following a further PBS 3 × 5 min wash, eye sections were isolated with a hydrophobic PAP pen (Vector Laboratories, Peterborough, UK). Non-specific antibody binding sites were blocked using 0.5% BSA, 0.3% Tween-20 and 15% Normal Goat Serum in PBS. Sections were then placed in primary antibody (1/200 rabbit anti-laminin antibody, L9393, Sigma; 1/200 rabbit anti-fibronectin antibody, F3648, Sigma; 1/100 goat anti-Brn3a antibody, SC-31984, Santa Cruz Biotechnology, Dallas, US) and left overnight at 4 °C before being washed 3 × 5 min in PBS and incubated for 1 h at room temperature with secondary antibody (1/400 goat anti-rabbit Alexa Fluor 594, A-11058, or 1/400 donkey anti-goat Alexa Fluor 488, A-11012, both from Thermo Fischer Scientific). Before mounting with Vectashield containing DAPI (Vector Laboratories), sections were washed 3 × 5 min in PBS. Control tissue sections, incubated without primary antibodies but with secondary antibodies, were all negatively stained (not shown).