Mouse anti nestin

Standard western blotting procedures were performed as described by Henderson et al.^{20 (link)}. The following antibodies were used: rabbit anti-CD51 (Abcam, Cambridge, MA, #112487, 1:1000); rabbit anti-p53 (Cell Signaling Technology, MA, #2527, 1:1000); rabbit anti-GAPDH (Cell Signaling Technology, MA, #2118 S, 1:1000); mouse anti-SP1 (Santa Cruz Biotechnology, CA, #sc-420, 1:500); mouse anti-SP3 (Santa Cruz Biotechnology, CA, #sc-136479, 1:500); mouse anti-Nestin (BD Biosciences, #611659, 1:1000); rabbit anti-Sox2 (Abcam, Cambridge, MA, #ab97959, 1:500)

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).

Western blot analyses were performed as previously described^{38 (link)}. Various antibodies, including rabbit monoclonal anti-REST (Abcam, Cambridge, UK), mouse anti-nestin (BD, San Jose, CA, USA), rabbit monoclonal anti-Pax-6 (Biolegend, San Diego, CA, USA), mouse monoclonal anti-β3-tubulin (Millipore, Billerica, MA, USA), mouse monoclonal anti-rhodopsin, rabbit polyclonal anti-recoverin (Millipore), mouse monoclonal anti-Brn3a (Millipore) and mouse monoclonal anti-Caspase-3 (Santa Cruz, California, USA) were diluted 1:1000, and mouse anti-β-actin (Sigma-Aldrich) was diluted 1:5000. The membranes were incubated with 1:5000 dilutions of DyLightTM680-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Sigma-Aldrich), and analyzed by Odyssey V 3.0 image scanning (LI-COR, Lincoln, NE, USA).

For immunofluorescence, cells were fixed with 4% paraformaldehyde and permeabilized in 0.3% Triton X-100. Antibodies included rabbit anti-TLX (1:1,000; Shi lab)45 (link), mouse anti-nestin (1:2,000; BD Pharmingen; Catalogue # 611659)55 (link), rabbit anti-integrin αv (1:500; Chemicon; Catalogue # AB1923)43 (link), mouse anti-neuropilin-1 (1:11; Miltenyi Biotec; Catalogue # 130-090-693)43 (link), mouse anti-GFAP (1:1,000; Sigma; Catalogue # G3893) and rabbit anti-Tuj1 (1:6,000; Covance; Catalogue # PRB-435P)56 (link).

Cultured CNPs at differentiation day 34 were dissociated in Accutase (ThermoFisher) for 10 min at 37 oC, then washed in PBS and counted. Samples containing 3*10^6 cells were fixed in 70% EtOH at − 20 °C overnight. The cells were washed three times in DPBS and blocked in solution 1%BSA-3% donkey serum for 45 min. Following Mouse anti-NESTIN (1/300, BD) antibody in 1%BSA-1% donkey serum or mouse anti IgG, 2 h at RT. Cells were washed in PBS three times and incubated in secondary antibody (alexa488 1:1000, Life technologies) in 1%BSA-1% donkey serum for 1 h at RT. Cells were washed twice in PBS and incubated with RNaseA (200 µg/ml, ThermoFisher) for 30 min. Then centrifuged and treated with DAPI (0.3 µg/ml, ThermoFisher) for 10 min. Cells were washed twice in DPBS and resuspended in 0.5 ml to be filtrated to remove clumps (Corning™ Falcon™ Test Tube with Cell Strainer Snap Cap). The samples were analysed on a BD LSRFortessa cell analyser (BD Biosciences). Data was analyzed in FlowJo (BD Biosciences) and Statistical analysis was performed in SPSS (IBM).