Brain Tissue Preservation and Handling

Mice were sacrificed following the completion of behavioral testing. This was conducted in one of two ways: (1) transcardial perfusion with PBS (Life Technologies, Carlsbad, CA, USA) followed by 4% paraformaldehyde (PFA; Sigma-Aldrich), post-fixation in 4% PFA overnight, and then storage in 20% sucrose until embedded in Tissue-Tek OCT compound (VWR, West Chester, PA, USA) and frozen at −80 °C, as previously described [20 (link),39 (link)]; (2) cervical dislocation and decapitation, followed by rapid extraction and flash freezing of the brain tissue in 2-methylbutane (Thermo Fisher Scientific, Asheville, NC, USA) placed in a dry ice and 70% EtOH bath, and storage at −80 °C until use for analysis below. In all cases, the brain and body masses of the mice were recorded.