Rabbit anti calretinin

Vibratome sections (60 μm thick) and retinal flat mounts were stained with rabbit anti-calretinin (1:1000, Millipore), goat anti-ChAT (1:500, Millipore), rabbit anti-recoverin (1:1000, Millipore), rabbit anti-PKARIIβ (1:500, BD Bioscience, San Jose, CA), rabbit anti-TH (1:1000, Millipore), rabbit anti-HCN4 (1:500, Neuromab, Davis, CA), rabbit anti-VIP (1:1000, Immunostar, Hudson, WI), mouse anti-melanopsin (1:1000, Advanced Targeting Systems, San Diego, CA), mouse anti-PKCα (1:500, Sigma, Saint Louis, MO), and mouse anti-Znp1/SytII (1:1000, ZIRC, Eugene, OR) for 1 (vibratome slices) or 5 days (flat mounts) at 4°C. The tissue was then washed in PBS (3 × 30 min), incubated with DyLight 405- (1:100, Jackson ImmunoResearch, West Grove, PA), Alexa Fluor 488-, Alexa Fluor 568-, and/or Alexa Fluor 633-conjugated secondary antibodies (1:1000, Invitrogen, Grand Island, NY) for 2 hr at RT (vibratome slices) or 2 days at 4°C (flat mounts), washed again in PBS (3 × 30 min), and mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) for confocal imaging.

Brains were fixed overnight at 4°C in 4% PFA in PBS, then cryoprotected in 30% sucrose in PBS for 1–3 days at 4°C, then frozen in OCT or cryogel on dry ice after 3–12 hr equilibration at 4°C. Sections were cut at 12 μm thickness spanning the rostral half of the SVZ (typically 6 sections per slide) or the entire olfactory bulb (typically 8 sections per slide). Sections were immunostained with the following primary antibodies: rat anti-BrdU (Abcam clone Bu1/75, 1/500, after heat-mediated antigen retrieval), guinea pig anti-Dcx (1/1000; Millipore), mouse anti-Mcm2 (BD Biosciences, 1/500, after heat-mediated antigen retrieval), rat anti-Ki67 (1/500; eBioscience), mouse anti-tyrosine hydroxylase (1/1000; Millipore), rabbit anti-calretinin (1/1000; Sigma), rabbit anti-calbindin (1/500; Millipore), rabbit anti-S100β (1/1000; Dako), rabbit anti-GST-pi (1/3000; Enzo, Farmingdale, NY), and mouse anti-NeuN (1/1000; Millipore). Fixed whole-mount SVZs were stained with mouse anti-acetylated tubulin (1/1000; Sigma), rabbit anti-β-catenin (1/500; Sigma), mouse anti-GFAP (1/3000; Sigma), and goat anti-EGFR (1/250; R&D Systems). Alexa Fluor 488-, 555-, and 647-conjugated secondary antibodies were used (Life Technologies, Carlsbad, CA).