title: A sensitive double immunostaining protocol for Fos-immunoreactive neurons.
abstract: 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.
authors.affiliation: Institute of Anatomy and Histology, University of Verona, Italy.
authors.forename: Z C
authors.initials: ZC
authors.lastname: Peng
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.forename: M
authors.initials: M
authors.lastname: Bentivoglio
mesh_terms: D000645:Ammonium Sulfate; D000818:Animals; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D009284:Naphthols; D009474:Neurons / Q000378:metabolism*; D009532:Nickel; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D011754:Pyronine / Q000031:analogs & derivatives; D051381:Rats; D017208:Rats, Wistar; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D013194:Staining and Labeling; D004124:p-Dimethylaminoazobenzene
chemical_list: D009284:Naphthols; D016760:Proto-Oncogene Proteins c-fos; D017475:Receptors, Nerve Growth Factor; C029350:1-naphthol; C031029:ammonium nickel sulfate; D009532:Nickel; D004124:p-Dimethylaminoazobenzene; C012020:pyronine B; D000645:Ammonium Sulfate; D011754:Pyronine