Immunocytochemical and stereological studies of the rat vestibular nucleus: optimal research methods using glucocorticoid receptors as an example

Many anatomists agree that stereological procedures for counting neurons are superior to simple density measurements because the latter are biased by the way that sections are sampled and the likelihood of selectively sampling neurons of particular sizes and orientations. Despite this, few stereological studies of the vestibular nucleus complex (VNC) have been conducted and all of the published immunocytochemical studies have used densitometry to quantify receptor numbers. One possible reason for this is the difficulty of combining stereological methods with immunocytochemical techniques required to label specific types of receptors. In this paper we detail the optimal methods for combining stereological and immunocytochemical techniques for the unbiased quantification of neuronal number, using glucocorticoid receptors as an example. We report that the cryofixation method is more effective than perfusion with paraformaldehyde, in order to obtain sections to estimate neuronal number using the Cavalieri and physical disector methods. Thionin proved to be the optimal counterstain for identifying both neurons and the boundaries of the VNC. The combination of stereological and immunocytochemical techniques offers an effective means to ensure accurate estimates of the number of neurons expressing a receptor.