A single observer, who had no knowledge of either the hormonal treatments being received or the odor stimulus being presented to individual subjects, used Observer 3.0 software (Noldus Co., Wageningen, Holland) to record the number of grid crossings and the duration of sniffing for each test session. Cheap Diskus Advair
These data were analyzed using a three-way ANOVA (sex X steroid treatment X olfactory stimulus), and post-hoc comparisons of group means were carried out using the Student-Neuman-Keuls procedure. The subjects from this behavioral study were later used in experiment 2.
The heart was exposed, and 1000 units of heparin (10 000 U/ml; Henry Schien, New York, NY) was injected into the left ventricle. Animals were perfused via the ascending aorta with approximately 50 ml of 0.1 M PBS, pH = 7.4, followed by 800 ml (for females) or 1000 ml (for males) of 4% paraformaldehyde in 0.1 m PBS at a pressure of 100 to 110 mm Hg. After perfusion, the brains were immediately removed and postfixed for 4 h in 4% paraformaldehyde. Brains were then cryoprotected in 30% sucrose/PBS at 4°C until they sank.
The right forebrain and olfactory bulb were frozen on dry ice and sectioned coronally at 30 ^m using a Reichert-Jung SM2000R table-top sliding microtome. Every brain section was saved in tissue antifreeze (500 ml 0.1 M Tris-buffered saline [TBS], 300 ml ethylene glycol, 300 g sucrose, 10 g polyvinylpyrrolidone (Mr 40 000) and distilled H2O to a volume of 1.0 L) and maintained at -20°C for later Fos or steroid receptor immunocytochemistry (ICC).