A triple-labeling method: HRP anterograde tract tracing combined with double immunofluorescent cell staining in developing neural tissue of the rat.

In this paper we describe a triple-labeling technique on a single section of developing nervous tissue. This labelling consists of a histochemical visualization of anterogradely transported horseradish peroxidase (HRP), within outgrowing rat corticospinal tract fibers and a double immunofluorescence labelling against glial fibrillary acidic protein (GFAP) and vimentin, being specific markers for astrocytes and their precursors, respectively. For the visualization of the anterogradely transported HRP, tetramethylbenzidine (TMB) is used as a chromogen with ammonium heptamolybdate (AHM) as a stabilizer followed by an additional diaminobenzidine-cobalt incubation to prevent washing out the HRP-TMB-AHM reaction product during the subsequent immunohistochemistry. Thereafter, double immunofluorescence labelling is carried out on the same section by adding simultaneously polyclonal anti-GFAP and monoclonal anti-vimentin which are then visualized with antisera conjugated with tetramethylrhodamine-isothiocynate (TRITC) and fluorescein-isothiocyanate (FITC) respectively as markers. Since the above mentioned visualization of the anterogradely transported HRP does not affect the immunofluorescence labelling, the methods enables within a single cryosection of developing neural tissue, the localization and identification of both outgrowing axons and their adjacent differentiating glial cells. In addition the method can easily be adapted for triple labelling of HRP tract tracing in conjunction with double immunofluorescence histochemistry of neurotransmitters.