Mouse anti brn3a

Fixed retinas were either frozen and sectioned at 20 μm in a cryostat or stained as whole mounts. Retina sections or whole mounts were incubated in PBS with 3% donkey serum and 0.3% Triton X-100 for blocking, followed by primary antibodies for ≥24 h at 4°C and secondary antibodies for ∼4 h. Retinas were then washed with PBS and mounted in Fluoromount G (Southern Biotech).
Primary antibodies used in this study were: rabbit anti-GFP (1:1000, Millipore); rabbit and mouse anti-calbindin (1:2000, Swant and 1:100, Swant); goat anti-choline acetyltransferase (ChAT), (1:400, Millipore); mouse anti-Brn3a (1:1000, Millipore); goat anti-Chx10 (1:200, Santa Cruz); mouse anti-AP2 (1:1000, DSHB); mouse anti-HCN4 (1: 1000, clone N114.10); rabbit anti-PKC alpha (1:1000, P4334 in Sigma); rabbit anti-secretagogin (1:4000, Biovendor); rabbit anti-arrestin (1:200, Milipore); rabbit anti-glutamine synthetase (1:1000, BD Biosciences); rabbit anti-sox9 (1:1000, Chemicon); mouse anti-connexin 43 (1:50, Life Sciences); rabbit antibody to Dab1 (a kind gift from B. Howell, SUNY Upstate Medical University). Secondary antibodies were conjugated to DyLight 649 (Jackson ImmunoResearch), Alexa Fluor 568, or Alexa Fluor 488 (Invitrogen) and used at 1:500.

The following primary antibodies were used: rabbit anti-Zic2 (RRID:AB_2315623, gift of Stephen Brown, 1:10,000), mouse anti-Islet1/2 (cat. # 39.4D5, RRID:AB_528173, gift of Thomas Jessell, 1:50; this antibody was raised against Islet1 but also reacts with Islet2), rabbit anti-cyclin D2 (Santa Cruz Biotechnology, Santa Cruz, CA, cat. # sc-452, RRID:AB_627350, 1:1000), rat anti-cyclin D2 (Santa Cruz Biotechnology, cat. # sc-593 RRID:AB_2070794, 1:50), goat anti-Brn3 (Santa Cruz Biotechnology, cat. # sc-6026 RRID:AB_673441, 1:200), and mouse anti-Brn3a (EMD Millipore, Billerica, MA, cat. # MAB1585 RRID:AB_94166, 1:200).
The following secondary antibodies were used: donkey anti-rabbit Alexa Fluor488 (Invitrogen, 1:400), donkey anti-mouse Alexa Fluor 488 (Invitrogen, 1:400), donkey anti-goat Alexa Fluor 488 (Invitrogen, 1:400), donkey anti-mouse Cy3 (Jackson Immunoresearch, West Grove, PA, 1:500), donkey anti-rabbit Cy3 (Jackson Immunoresearch, 1:500), donkey anti-goat Cy5 (Jackson Immunoresearch, 1:200), and donkey anti-rat Cy3 (Jackson Immunoresearch, 1:500).

Immunostainings for Brn3a, GFP and RBPMS on retinal whole-mounts were completed as described previously^{22 (link),28 (link),29 (link)}. The primary antibodies used are: mouse anti-Brn3a for single labelling (1:500, Merck Millipore, Darmstadt, Germany); goat anti-Brn3a for co-labelling with GFP or RBPMS (1:750, Santa Cruz, Dallas, TX, USA); rabbit anti-GFP (1:10,000, in-house made); and rabbit anti-RBPMS (1:200, PhosphoSolution, Aurora, CO, USA). Immunolabeled RGCs were examined under confocal microscopy (LSM 710, Carl Zeiss MicroImaging GmbH, Jena, Germany). Each retinal whole mount was imaged as a tiled z-stack at × 10 magnification, which was used to generate a single plane maximum projection of the RGC layer in each retina for subsequent analysis. Retinal image acquisition settings were kept constant for all retinas imaged, allowing comparison of Brn3a expression in each experimental group as previously described³⁰ . Each whole mount image was manually orientated, using in vivo cSLO imaging of retinal vasculature as a reference, so that the superior retina was towards the top of the image.