Mouse anti brn3c

After electrophysiological experiments, some retinas were fixed in fresh 4% paraformaldehyde in PBS at 4 degrees C for 1 hour. After fixation, the retinas were washed and incubated at 4 degrees C with primary antibodies for 4–5 days. Secondary antibody incubation at room temperature for at least 2 hours preceded mounting on a glass slide with spacers, ganglion cell side up, with Prolong Gold (Invitrogen). Whole mount images were obtained on a LSM 710 inverted NLO microscope at 20X or 40X (Zeiss). The primary antibodies used were: anti-GFP (rabbit, Life Technologies; chick, Abcam); mouse anti-Nonphosphoneurofilament H (SMI-32, Covance); goat anti-Osteopontin (R&D Systems); rabbit anti-Parvalbumin, rabbit anti-Calbindin, and mouse anti-Calretinin (all from Swant); anti-vAChT (goat, Promega; guinea pig, Millipore); goat anti-ChAT (Millipore); mouse anti-Brn3a (Millipore); goat anti-Brn3 (raised against Brn3b, Santa Cruz Biotechnology) and mouse anti-Brn3c (Santa Cruz Biotechnology). Dylight405-, Alexa488-, Cy3- and Alexa647-conjugated secondary antibodies were obtained from Jackson Immunoresearch.

Rabbit anti-WHRN (from Joriene de Nooij lab), mouse anti-ISLET1 (DSHB, cat#39.4D5), chicken anti-RFP (Rockland, cat#600-901-379S), rabbit anti-VGLUT1 (SYSY, cat#135303), guinea pig anti-VGLUT1 (SYSY, cat#135304), rabbit anti-FXYD7 (Sigma-Aldrich, cat#HPA026916), rabbit anti-LMCD1 (Human Protein Atlas, cat#HPA024059), goat anti-WGA (Vector Laboratories, cat#AS-2024), mouse anti-BRN3C (Santa Cruz Biotechnology, cat#sc-81980), goat anti-PV (Swant, cat#PVG-213), rabbit anti-PV (Swant, cat#PV27), rabbit anti-CART (from Igor Adameyko lab), rabbit anti-RUNX1 (from Thomas Jessell lab), rabbit anti-CALB1 (Swant, cat#CB-38a), goat anti-CHAT (Millipore, cat#AB144p), DAPI (Invitrogen, cat#D1306)

For immunostainings, retinas were fixed in 4% PFA and transferred to a permeabilizing and blocking solution containing 0.3% Triton X-100, 5% donkey serum, 0.2% bovine serum albumin, 0.2% lysine, and 0.2% glycine for at least 24 h. After three washes in PBS, retinas were incubated with one of the following primary antibodies (all antibodies were used at a dilution of 1:100, unless otherwise stated): guinea pig anti-RBPMS (ABN1376, Millipore, United States); rabbit anti-Calbindin-D-28k 1:200 (702411, Thermo Fisher Scientific, United States); rabbit anti-CART 1:300 (14547S, Cell Signaling Technology, United States); rabbit anti-ChAT (AB-N34AP, ATS, United States); rabbit anti-Parvalbumin (PV27, Swant, Switzerland); rabbit anti-Satb1 (ab70004 abcam); mouse anti-SMI32 (801701, BioLegend, United States); mouse anti-Brn3c (sc-81980, Santa Cruz Biotechnology, United States). The tissue was incubated for 1–3 days with the primary antibodies (in 0.3% Triton X-100, 2% bovine serum albumin, 0.02% sodium azide in PBS). After 3 washes in PBS, the secondary antibody (in 0.3% Triton X-100, 3% donkey serum in PBS) was added for at least 1 day.