A SENSITIVE DOUBLE IMMUNOSTAINING PROTOCOL FOR FOS-IMMUNOREACTIVE NEURONS

Immunostaining of Fos, the nuclear protein encoded by the immediate ea rly gene c-fos, is widely used to reveal the functional activation of neurons. The chemical identity of cells that express c-fos can be inve stigated with double immunohistochemistry. We report the usefulness of a sequential two-color avidin-biotin-immunoperoxidase method that pro vides a highly sensitive double immunostaining and allows long-term st orage of the sections. In this protocol, metal intensification of diam inobenzidine (int-DAB) resulted in dark brown/black Fos immunostaining of the neuronal nucleus. The use of alpha-naphthol/pyronin reaction p roduct yielded pink immunostaining of a second antigen in the cytoplas m. This combination produced higher contrast than that produced by int -DAB Fos immunostaining combined with conventional DAB light brown cyt oplasmic staining. The sensitivity of the use of int-DAB and alpha-nap hthol/pyronin was verified in different experimental paradigms, combin ing the immunocytochemical detection of Fos with that of the p75 nerve growth factor receptor, or parvalbumin, or calbindin D28k.