2.10 Brain tissue collection

Rats were killed by rapid decapitation and the brains were isolated, and dissected along the midsagittal plane. The left hemisphere was snap frozen for Western blotting analysis (see below) and the right hemisphere was postfixed in 4% paraformaldehyde for immunohistochemistry. For tissue fixation, the hemispheres were incubated at room temperature for 36 hours and subsequently at 4 °C for 48 hours with fresh paraformaldehyde replacing the old solution every 12 hours. Finally, the hemispheres were transferred to sucrose solution (30% sucrose with 0.1% sodium azide) for cryoprotection and storage till tissue sectioning was conducted (Cohen et al., 2015). Subsequently, the tissue was sliced in 40µm sections along the coronal plane on a freezing microtome. Every ninth section through the PFC (+3.7 to +2.5 mm from bregma; 4 sections per rat) was mounted on Superfrost® Plus slides and dried overnight and used for BrdU analysis. Two sections through the PFC (+3.2 and +2.7 mm from bregma) were mounted as described before and processed for cFos analysis.