Brain Tissue Fixation and Cryoprotection

Ninety minutes after the last behavioral test, animals were deeply anesthetized and perfused through the heart with 100 mL of 0.1 M PBS solution (phosphate-buffered solution; 5.52 g of NaH2PO4 + 21.88 g of Na2HPO4), followed by 100 mL of 4% formaldehyde (diluted in PBS solution). The brains were removed and post-fixed in a 4% formaldehyde solution followed by a hypertonic solution of sucrose 30% (diluted in PBS) for cryoprotection. The brains remained in this solution until they showed signs of dehydration, a procedure that usually occurs within 24 h. Immediately after this period, the brains were dried and frozen at -80 ºC. Subsequently, the brains were sectioned in a cryostat (Leica CM1850) in coronal brain sections (50 µm thick) at intervals of three slices. For every three sections, one was selected (for immunohistochemistry processing), and two were discarded. Then, the sections were stored in an antifreeze solution (300 g of sucrose, 500 mL of PBS solution, and 300 mL of ethylene glycol) at -20 ºC until immunohistochemical processing.