Rabbit anti s 100β

Detailed procedures regarding tissue processing and antibody staining, including BrdU detection, were previously described (Suh et al., 2007 (link)). Primary antibodies used were mouse anti-Neuronal Nuclei (NeuN 1:10; kindly provided by Dr. R. Mullen, University of Utah); mouse anti-Nestin (1:500; Pharmingen); goat anti-Doublecortin (DCX 1:200; Santa Cruz Biotechnologies); rabbit anti-Glial Fibrillary Acidic Protein (GFAP 1:1000; Dako); rabbit anti-S-100β (1:5000; Swant); rat anti-BrdU (1:200; Accurate Chemicals); rabbit anti-Ki67 (1:200; Novocastra); rabbit anti-Sox2 (1:200, Chemicon); rabbit anti-brain lipid binding protein (BLBP 1:1000; kindly provided by N. Heintz, Rockefeller); rat anti-MUSASHI-1 (1:1000; a kind gift from O. Hideyuki, Keiyo University, Japan); rabbit anti-GFP (1:100, Molecular Probes); and guinea pig anti-GFAP (1:1,000). Fluorescence immunohistochemistry (IHC) was performed using corresponding FITC, Cy3, or Cy5 secondary antibodies (1:200, all raised in donkey, Jackson ImmunoResearch, West Grove, PA, United States). DAPI (10 mg/ml, Sigma) was used as a fluorescent counterstain.
Confocal stack images of brain slices (40 μm) were obtained with the Confocal A1 Nikon Inverted SFC with 40× objective and the Zeiss Spinning Disk with a 20× objective. Cell quantification and analysis was performed using NIS-Elements software (Nikon) and Zen Blue (Zeiss).

Sections were processed for DNA denaturation as detailed above followed by several TBS rinses and incubation in TBS++ for 30 min. Primary antibodies were used as follows: sheep anti-BrdU (1 : 800; Biodesign, Saco, ME), rabbit anti-S100β (1 : 500, Swant, Bellinzona, Switzerland), and rabbit anti-NeuN (neuronal nuclei; 1 : 500, Millipore, Billerica, MA). Sections were incubated with primary antibodies for 48 hrs at +4°C. After several rinses in TBS++ for 10 min at room temperature, the secondary antibodies coupled to fluorescein isothiocyanate (FITC), cyanine 3 (CY3), and cyanine 5 (CY5) were applied for 2 hrs (all used at 1 : 250 in TBS++; Jackson ImmunoResearch, West Grove, PA). Sections were washed three times in TBS and coverslipped in 20% polyvinyl alcohol (average MW 30,000–70,000) in 50% glycerol (w/v) containing 2.5% w/v 1,4-diazabicyclo-[2.2.2]-octane (Sigma).