For MRI and IHC studies, mice were first anesthetized with pentobarbital (100mg/kg, i.p.) and then sacrificed by perfusion as described previously (Coleman et al., 2009; Crews et al., 2004). Briefly, mice were perfused with 0.1M phosphate buffered saline (PBS) followed by 4% PFA followed by post-fixation in 4% PFA at 4°C for 24 hours. The ears and skin were then removed from the head and the skull was re-immersed in 4% PFA for 24 hours. Mouse skulls with the brains intact were then submerged in a 1% PFA/PBS solution at 4°C until MRI. To ensure that time between imaging did not alter brain volume; four postmortem adult mice were scanned twice with each scan being three months apart. Importantly, there was no significant difference in total brain volume across this 3 month time period in these mice (t-test, p=0.62). Neither did we observe a correlation between brain volume and scan date (R= −0.0017, p=0.994).
