Brain Histology Preparation and Imaging

Brain histological sections were prepared using a protocol modified from our previous studies (Jeffrey et al., 2014 (link); Stover et al., 2017 (link)). Mice were anesthetized via sodium pentobarbital (100 mg/kg, intra-peritoneal injection) and perfused trans-cardiacally with saline and then with 10% neutral buffered formalin solution (Sigma-Aldrich; Oakville, Ontario, Canada). Removed brains were further fixed in a hypertonic (with 20% sucrose) formalin solution. Coronal sections of 50 μm thick were obtained using a Leica CM3050 research cryostat and placed onto glass slides (Superfrost plus microscope slides, Fisher Scientific, Canada). Brain sections were dried in room air for ≥1 week, processed sequentially with chloroform (24 h), 95% ethanol (24 h), 90% ethanol (12 h), and 70% ethanol (0.5 h), and then stained with cresyl violet (0.1%, Sigma Aldrich, Oakville, Ontario, Canada). Images of brain sections were obtained using a slide scanner (Aperio digital pathology slide scanner AT2, Leica) at 20 × magnification and analyzed using ImageScope (Leica) or Image J (National Institute of Health, USA) software.