Slabs from the frozen brains were serially cryosectioned at 14 µm onto PEN slides for LMD (Leica Microsystems, Inc., Bannockburn, IL) and a 1:10 Nissl series was generated for neuroanatomical reference. After drying for 30 min at room temperature, PEN slides were frozen at −80°C. Slides were later rapidly fixed in ice cold 70% ethanol, lightly stained with cresyl violet to allow cytoarchitectural visualization, dehydrated, and frozen at −80°C. LMD was performed on a Leica LMD6000 (Leica Microsystems, Inc.) using the cresyl violet stain to identify target brain regions. Samples captured include cortical and subcortical regions and are listed for each brain in the ontological sample map (Suppl. Table 2).
