Mouse anti cnp

Cells were fixed in 4% paraformaldehyde, rinsed and then permeabilized in phosphate buffered saline (PBS) containing 5% normal goat serum (NGS) and 0.4% Triton-X100 for 1 h at room temperature (RT). For BrdU immunostaining, cells were preincubated in 2N HCl at RT for 20 min, followed by neutralization with 0.1 M sodium borate buffer (pH 8.5) prior to immunolabeling. Cells were then incubated overnight at 4 °C in PBS containing 3% NGS, 0.1% Triton X-100 and primary antibody as follows; guinea pig anti-NG2 (1:300); rabbit anti-platelet derived growth factor α receptor (PDGFαR) (1:600, both courtesy of Dr. Bill Stallcup); rabbit anti-Olig2 (1:1000, Millipore); mouse anti-MBP (smi99, 1:1000, Covance); rat anti-BrdU (1:100, Accurate); mouse anti-CNP (1:750, Millipore) and chicken anti-GFP (1:1000, AbCam). The next day, cells were rinsed and incubated for 1 h at RT with species-specific secondary antibodies directly conjugated to Alexa fluorophores (1:1000, Life Technologies). Coverslips were mounted in ProLong® Gold anti-fade reagent with 4',6-diamidino-2-phenylindole (DAPI) and visualized on a Zeiss Axio Observer Z1 epifluorescence microscope. Images were captured and processed using AxioVision 4.8 software. Cell counts were made at 200× from three fields of view per condition in three separate experiments.