Androgen receptor (AR) and estrogen receptor (ER) ICC were performed on alternate sections of olfactory bulb and forebrain from a subset of male and female ferrets used in experiment 1. Androgen receptor ICC was performed according to the protocol published by Kashon et al. for ferret brain, using brain sections from two gonadectomized, TP-treated females and males in addition to one gonadally intact breeding male. Brain sections were preincubated with 0.5 M glycine in 0.1 M PBS for 30 min and blocked with 0.5% H2O2/4% normal goat serum in 0.1 M PBS for 30 min. Sections were then incubated in primary PG-21 AR antiserum (1 ^g/ml) for 48 h at 4°C, and biotinylated goat anti-rabbit secondary antibody (1:200; Vector Labs.) for 4 h followed by ABC (1:100) for 2 h; they were then reacted with nickel DAB solution for 4 min. ventolin 100 mcg
Estrogen receptor ICC was performed on brain sections from three females and two males that had been gonadectomized and treated with oil vehicle. Brain sections were prewashed in 0.05 M sodium meta-periodate for 15 min and sodium borohydrate for 15 min. Sections were then incubated in primary H222 ER antibody (2 ^g/ml), for 36 h at 4°C followed by biotinylated sheep anti-rat secondary antibody 1:200 (Amersham Life Science, Piscataway, NJ) for 2 h and ABC (1:100) for 2 h; sections were then reacted with nickel DAB solution for 7 min. We used gonadectomized TP-treated subjects for AR ICC and oil-treated subjects for ER ICC in order to maximize the likelihood of seeing any AR or ER immunoreactive cells in ferrets’ olfactory bulbs.