Rabbit anti pax2

Protein isolation and Western blots were performed as previously described [68 (link)]. Briefly, cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS supplemented with protease inhibitors (Complete, Roche; 1 mM PMSF, Sigma-Aldrich; 1mM sodium orthovandate, Sigma-Aldrich). Total protein in the extracts was quantitated using a colorimetric BCA Protein Assay Kit as per the manufacturer's instructions. 40 μg of total cellular protein for each sample was boiled in Laemmli sample buffer and separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membrane (Hybond-C Extra). Immunoreactive bands were visualized by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (containing equal parts of the Stable Peroxide Solution and the Luminol/Enhancer Solution) (Thermo Scientific, USA). Antibodies used for Western blotting were rabbit anti-PAX2 (1:500; Zymed), mouse anti-SMAD2/3 (1:1000; Santa Cruz Biotechnology), goat anti-GAPDH (1:2000, Santa Cruz Biotechnology), and anti-rabbit and anti-mouse horseradish peroxidase IgG (Sigma-Aldrich). The protein ladder used was MagicMark XP Standard (Invitrogen, USA).

Primary antibodies used included the following: Guinea pig anti-VGLUT3 (1:10,000, Millipore), sheep anti-tyrosine hydroxylase (1:500, Millipore), rabbit anti-melanopsin (1:10,000, ATS), mouse anti-Brn3a (1:500, Millipore), goat anti-choline acetyltransferase (1:500, Millipore), mouse anti-NeuN (1:500, Millipore), mouse anti-GFAP (1:500, Sigma), rabbit anti-FoxP2 (1:4000, Abcam), rabbit anti-Pax2 (1:200, Zymed), rabbit anti-cleaved caspase 3 (1:100, Cell Signaling Technologies), mouse anti-parvalbumin (1:500, Sigma), and mouse anti-GAPDH (1:500, Abcam). Mouse monoclonal antibodies against γ-Pcdh proteins used for western blots (1:500–1:1000) were generated by NeuroMab in collaboration with the Weiner laboratory [55 (link)] and obtained from Antibodies, Inc.: N159/5 (detecting an epitope in constant exon 1 or 2 and thus all 22 γ-Pcdh isoforms); N144/32 (detecting all γA subfamily isoforms); N148/30 (specific for γB2); N174B/27 (specific for γC3). A rabbit polyclonal antibody raised at Affinity BioReagents against the peptide sequence VAGEVNQRHFRVDLD (within EC1) from murine γC4 was also used for western blotting (1:1000). Secondary antibodies were conjugated with Alexa-488, -568, or -647 (1:500, Invitrogen) or HRP (1:1000–1:5000, Jackson Immunoresearch).