Mouse anti brn3a

Histological and immunofluorescence staining on cryosections was performed as described previously (de Melo et al., 2005 (link)). Primary antibodies used were: mouse anti-BrdU (1:200, Chemicon), mouse anti-BRN3A (1:200, Santa Cruz), goat anti-BRN3B (1:200, Santa Cruz), rabbit anti-caspase 3 (1:500, Cell Signaling Technologies), rabbit anti-DLX2 (1:400, C199 affinity purified), mouse anti-ISLET1 (1:600, DSHB, University of Iowa), rabbit anti-phosphohistone H3 (1:1000, Upstate), rabbit anti-PROX1 (1:500, Chemicon), rabbit anti-PAX6 (1:800, Covance) and mouse anti-syntaxin (1:6000, Sigma). Secondary antibodies and fluorescent tertiary molecules used were: FITC-conjugated goat anti-rabbit (1:200), biotin-SP-conjugated goat anti-rabbit (1:200), biotin-SP-conjugated goat anti-mouse (1:200) (Jackson ImmunoResearch), streptavidin-conjugated Oregon Green 488 (1:200) and streptavidin-conjugated Texas Red (1:200) (Molecular Probes). Negative controls omitted the primary antibody. TUNEL staining used the In Situ Cell Death Detection Kit, TMR Red (Roche Diagnostics). Non-radioactive digoxigenin in situ RNA hybridization was performed as described previously (de Melo et al., 2005 (link)).

Retinas from both eyes were dissected and processed as described (15 (link)). Briefly, retinas were postfixed for 1 to 3 hours in 4% PFA, cryoprotected in 30% sucrose solution overnight at 4°C, embedded using OCT, and rapidly frozen on dry ice. Sections of 15 μm were sliced on a cryostat and collected directly onto microscope slides and stained. Primary antibody (mouse anti-Brn3a, Santa Cruz Biotechnology, 14A6, sc-8429) was applied overnight at 4°C. The secondary antibody used was Alexa Fluor 488–conjugated goat anti–mouse IgG (Invitrogen, A11001). TO-PRO-3 iodide (642/661) was used to stain nuclei. Fluorescent images were scanned using a Zeiss LSM710 Spectral confocal microscope with a 40× objective and processed with Zen 2.1 software (Carl Zeiss). Within the ganglion cell layer, the number of immunostained Brn3a cells was counted per 100 mm for each retinal section from the central regions. Data were averaged from 3 slices of each retina.