Mouse monoclonal anti brn3a

Western blot analyses were performed as previously described^{38 (link)}. Various antibodies, including rabbit monoclonal anti-REST (Abcam, Cambridge, UK), mouse anti-nestin (BD, San Jose, CA, USA), rabbit monoclonal anti-Pax-6 (Biolegend, San Diego, CA, USA), mouse monoclonal anti-β3-tubulin (Millipore, Billerica, MA, USA), mouse monoclonal anti-rhodopsin, rabbit polyclonal anti-recoverin (Millipore), mouse monoclonal anti-Brn3a (Millipore) and mouse monoclonal anti-Caspase-3 (Santa Cruz, California, USA) were diluted 1:1000, and mouse anti-β-actin (Sigma-Aldrich) was diluted 1:5000. The membranes were incubated with 1:5000 dilutions of DyLightTM680-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Sigma-Aldrich), and analyzed by Odyssey V 3.0 image scanning (LI-COR, Lincoln, NE, USA).

Samples were fixed with 4% PFA for 10 min at room temperature, subsequently washed with PBS, permeabilized, and blocked in blocking solution (with 2% BSA, 0.1% Triton-X100 in PBS) for 2 h. Primary antibodies were added at 1:100 dilution [rabbit polyclonal anti-MyoVIIa (Proteus); mouse monoclonal anti-Sox2 (Millipore); rabbit polyclonal anti-Sox2 (Invitrogen); rat anti-E-cadherin (Abcam); mouse anti-GATA3 (Thermo Fisher Scientific); mouse anti-Islet 1 (DSHB, deposited by Jessell T.M.); goat anti-Doublecortin (Santa Cruz Biotechnology); rabbit anti-Pax2 (Thermo Fisher Scientific); rabbit anti-Pax8 (Abcam); mouse anti-Nestin (BD Transduction Laboratories); mouse anti-βIII-Tubulin (R&D); rabbit anti-Peripherin (Millipore); and mouse monoclonal anti-Brn3a (Millipore)], and incubated in blocking solution overnight at 4°C. Samples were then washed three times with PBS, followed by the addition of Alexa Fluor conjugated secondary antibodies (Invitrogen) at 1:500 dilution in blocking buffer for 2 days at room temperature. The images were acquired with a confocal microscope (Zeiss LSM 700) using 10× and 20× air objectives.