Sex Difference and Testosterone Modulation: MATERIALS AND METHODS(6)

11 Mar


Fos ICC was performed on every fourth section from the forebrain and olfactory bulb. One animal from each treatment group was included in each ICC run. Free-floating sections were incubated in 3% normal goat serum/1% H2O2/PBS for 1.5 h at room temperature and then transferred to primary Fos antiserum (DCH-1; 1:5000 in 0.4% Triton X-100/PBS; rabbit polyclonal antiserum raised against N-terminal amino-acids 2-17). Sections were incubated in Fos primary antiserum on a shaker for 16 h at room temperature. Sections were then rinsed four times with 0.1 M PBS at room temperature for 10 min, incubated with biotinylated goat anti-rabbit antibody (1:200; Vector Labs., Burlingame, CA) for 2 h, washed four times in 0.1 M PBS, incubated for 2 h in ABC (1:100; Vector Labs.), and washed 4 more times in 0.1 M PBS. proventil inhaler

Sections were then reacted with nickel diaminobenzidine (DAB) solution (Vector Labs.) for 7 min and washed 5 times in 0.1 M PBS. Sections were mounted on gelatin-coated slides and air-dried before being coverslipped with Permount (Fisher Scientific Co., Pittsburgh, PA). The specificity of the DCH-1 Fos-antibody in ferret brain has been demonstrated previously.