Chicken anti nestin

For immunocytochemistry, cells were cultured on glass coverslips. After washing in PBS, cells were fixed 10 min at RT with PFA 4%. For Rnd2 staining, we used the PGT-based protocol described in “Immunohistochemistry section” with a fluorescent secondary antibody. For other stainings, cells were treated with PBS – 0.01% Triton X-100 – 1% bovine serum albumin (BSA, Sigma) for 30 min and incubated overnight at 4 °C with primary antibodies diluted in blocking solution: mouse anti-MAP2 (1/500, Sigma, M4403), chicken anti-nestin (1/400, Aves Labs, NES), mouse anti-Tuj1 (1/2000, Promega, G712A). Cells were then incubated with appropriate fluorescent secondary antibodies. Following this step, DAPI was added for 10 min. Images were acquired using an Eclipse Ti-U Nikon or a Leica SP5 microscope.

As described previously (Palmer et al., 1999 (link)), cells on coverslips were rinsed in PBS and fixed with 4% PFA in 0.1 M phosphate buffer at room temperature for 15 min. After a 1-h pre-incubation in blocking solution (10% donkey serum and 0.2% Triton X-100 in PBS), the cells were incubated with primary antibodies at 4°C overnight, and at room temperature for 2 h with Alexa Fluor-conjugated secondary antibodies (1:500, Invitrogen). For BrdU staining, the cells were pretreated with 2 N HCl for 30 min at 37°C before blocking. The following primary antibodies were used: rat anti-BrdU (1:1000) and chicken anti-Nestin (1:2000, Aves Labs, Tigard, OR, USA).

Neurospheres were placed into poly-D-lysine-coated glass-bottom dishes (Mattek) and allowed to attach for 6 hours while in a thin film of SCM, DMEM with 10% fetal bovine serum (FBS) and P/S (SM), or DMEM with B27M (Life Technologies, Grand Island, NY, USA) and P/S (B27M). After the neurospheres attached, 2 ml of medium (SCM, B27M or SM) was added to prevent loss of neurospheres. Neurospheres were fixed in 100% methanol for 10 minutes and standard immunocytochemistry was performed. Immunofluorescence staining was used to identify neural stem progenitor cells, neural progenitor cells, neurons, and astrocytes. Primary antibodies were used at the following dilutions: chicken anti-NeuN (Aves Labs, Tigard, OR, USA) 1:1000; chicken anti-Nestin (Aves Labs) 1:1000; rabbit anti-BetaIII-tubulin (Cell Signaling Technology, Danvers, MA, USA) 1:1000; rabbit anti-Musashi1 (Msi1, Cell Signaling Technology) 1:1000; rabbit anti-GFAP (Cell Signaling Technology) 1:1500; rabbit anti-SOX2 (Life Technologies) 1:500; cleaved caspase–3 (Cell Signaling Technology) 1:500. Samples were rinsed after overnight incubation at 4°C, and were incubated for 2 hours with appropriate Alexa488 and 458-conjugated secondary antibody (Life Technologies). Confocal microscopy of spheres was performed as mentioned in our previous study [35 (link)].