For BrdU immunocytochemistry, DRG NSCs were permeablized with 1% Tx-100 in PBS for 30 min followed by 2 N HCl in 0.05 M PBS at 37°C and neutralization with 0.1 M borate buffer. The endogenous peroxidase activity was quenched with 3% H2O2 in 10% methanol. Nonspecific binding was blocked with 10% normal serum from the secondary antibody host species. Primary antibody was incubated in 0.5% Tween-20 + 2% normal sheep serum in PBS was applied overnight. Fluorescein-isothiocyanate (FITC)-conjugated secondary was used for fluorescent detection. Negative controls for immunostaining included omission of primary antibody and incubation with preimmune serum from the same species used to produce the secondary antibody. The immunostaining was performed in triplicate. The clonal analysis and responsiveness to EGF/bFGF experiments were performed in duplicates. Slides were viewed using a Leitz Orthoplan II microscope using bright field if DAB was the chromagen. Fluorescent labeling was viewed using the corresponding FITC, rhodamine, or UV filter. Photographs were taken with a Spot II camera and image acquisition software.
