Rat Brain Fixation and Imaging

All protocols were approved by the Loma Linda University Animal Health and Safety Committee and are in compliance with federal regulations (protocol #813033, approved 09/17/2013). All experiments were conducted following the ARRIVE guidelines. Male adult Sprague Dawley rats (329.8 ± 3.3 g, Harlan; n = 24) recovered in the vivarium for 5–7 days prior to transcardiac perfusion using 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA, USA). Though imaging of either the brain in the cranial vault [30 (link)] or of the brain alone has been reported, brain-only samples have been used to generate atlases [31 (link)]. Therefore, we elected to remove the brains from the cranial vault. Brains were postfixed in 4% PFA, washed and stored at 4 °C in 0.1M PBS/0.05% sodium azide until imaging and histology. Together, these procedures reduced the potential for artifacts particularly at high field strengths. Prior to imaging, brains were placed in Fluorinert (FC-770, SynQuest Labs, Inc., Alachua, FL, USA) to facilitate susceptibility matched imaging.