Double c-Fos/RFRP-3 and AVP/RFRP-3 immunohistochemistry
This protocol is extracted from research article:
Daily and Estral Regulation of RFRP-3 Neurons in the Female Mice
J Circadian Rhythms, Apr 15, 2021; DOI: 10.5334/jcr.212

The number of c-Fos expressing RFRP-3 neurons and the density of AVP fibers surrounding RFRP-3 neurons were assessed by dual immunohistochemistry. Brain sections were first incubated either with a rabbit polyclonal antiserum raised against c-Fos (1:2000; Santa Cruz Biotechnology; RRID: AB_627251) or a rabbit polyclonal antiserum raised against neurophysin II, a cleavage product of prepro-vasopressin (1:15000; Sigma-Aldrich; RRID: AB_260747) diluted in 154 mM PBS buffer containing 10% donkey serum and 0.3% Tween 20, for 24 hours at room temperature. Brain sections were washed with PBS, incubated with biotinylated donkey antirabbit (1:2000; Jackson Labs; in 154 mM PBS buffer containing 10% donkey serum and 0.3% Tween 20) for 1 hour and then washed again with PBS. Immunoreactive signal was amplified by a treatment with the avidin biotin complex coupled to peroxidase (1:250; Vector Laboratories) for 1 hour, then revealed using a solution of 0.5 mg/mL 3,3-diaminobenzidine (DAB; Sigma-Aldrich) diluted in water and 0.001% hydrogen peroxide urea (Sigma-Aldrich) for 30 minutes. Before performing the second immunolabelling, the antibodies were eluted with 2 x 15 minute washes in a solution of 100 mM glycine containing 0.3% Triton X-100 (pH 2.2). Brain sections were then incubated with a primary antibody directed against RFRP-3 (1:15000; rabbit anti-RFRP-3, University of Otago, NZ; RRID: AB_2877670; in a PBS buffer containing 10% donkey serum and 0.3% Tween 20) overnight at room temperature. The sections were then washed with PBS, incubated with biotinylated donkey anti-rabbit (1:2000; Jackson Labs; in 154 mM PBS buffer containing 10% donkey serum and 0.3% Tween 20) for 1 hour, and then washed again with PBS. The RFRP-3 signal was detected using streptavidin-peroxidase at 1/3000 (Roche) for 1 hour and revealed with Fast blue- BB (Sigma-Aldrich) for 7 minutes. Finally, sections were mounted with CC/mount (Sigma-Aldrich), dehydrated in toluene twice for 10 minutes, and mounted with Eukitt resin (Sigma-Aldrich).

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