Immunocytochemistry of Neural Progenitor Cells

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)].