A sensitive double immunostaining protocol for Fos-immunoreactive neurons

Immunostaining of Fos, the nuclear protein encoded by the immediate early gene c-fos, is widely used to reveal the functional activation of neurons. The chemical identity of cells that express c-fos can be investigated with double immunohistochemistry. We report the usefulness of a sequential two-color avidin-biotin-immunoperoxidase method that provides a highly sensitive double immunostaining and allows long-term storage of the sections. In this protocol, metal intensification of diaminobenzidine (int-DAB) resulted in dark brown/black Fos immunostaining of the neuronal nucleus. The use of alpha-naphthol/pyronin reaction product yielded pink immunostaining of a second antigen in the cytoplasm. This combination produced higher contrast than that produced by int-DAB Fos immunostaining combined with conventional DAB light brown cytoplasmic staining. The sensitivity of the use of int-DAB and alpha-naphthol/pyronin was verified in different experimental paradigms, combining the immunocytochemical detection of Fos with that of the p75 nerve growth factor receptor, or parvalbumin, or calbindin D28k.