consisted of 10 ng/ml NGF or BDNF; controls received an equal volume of medium. After 28 days, cells were fixed with 4% formaldehyde for immunostaining for TrpV1 expression for subsequent counting (described below). Because BDNF influences neuronal maturation, we investigated whether the proportion of mature neurons, detected with an antibody against mature neuronal nuclear protein (NeuN), increased in BDNF-treated aDRG NSCs. All TrpV1-immunostained, NeuN-immunostained cells, and Nissl-stained cells were counted on a Leitz Orthoplan2 microscope under a 25× objective. Inclusion criteria were as follows: TrpV1-immunostained cells must have punctuate-like brown DAB staining on the plasma membrane and NeuN-immunostained cells contained nuclear staining, in some cases filling the cytoplasm.
