Transcardial Perfusion and Brain Fixation

Under anesthesia induced via intraperitoneal administration of a mixture of medetomidine (0.3 mg/kg BW, Domitor; ZENOAQ, Koriyama, Japan), Midazolam (4 mg/kg BW, Midazolam; SANDOZ, Tokyo, Japan), and butorphanol (5 mg/kg BW, Bettlefar; Meiji Seika Pharma, Tokyo, Japan), rats were perfused with 100 ml of 0.2% heparinized 0.1 M phosphate buffer (PB; pH 7.4), followed by 1,000 ml of 3% paraformaldehyde in 0.1 M PB through the ascending aorta, as previously reported (Oda et al., 2010 (link), 2018 (link)). In our experience, fixation with 3% paraformaldehyde occasionally leads to improved preservation of immunoreactivity for G-protein coupled receptors such as dopamine receptors, with structural preservation comparable to that of 4% paraformaldehyde. Following perfusion, the brains were removed and postfixed for 3 h in 3% paraformaldehyde in 0.1 M PB. Blocks of tissue specimens were immersed in 20% sucrose in 0.1 M PB overnight and then sectioned at a thickness of 60 μm with a freezing microtome. The sections were stored in tissue cryoprotective solution (25% glycerol and 30% ethylene glycol in 0.05 M PB) at −80°C until use.