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.