Aperio digital pathology slide scanner at2

Brain histological assessments were performed as previously described (Bin et al., 2018 (link); Song et al., 2018a (link)). Briefly, the mouse was anesthetized by sodium pentobarbital and transcardially infused with the dissection-only ACSF and then with 10% neutral buffered formalin solution. The brain was removed and further fixed in a hypertonic (with 20% sucrose) formalin solution for ≥24 h. Cryostat coronal sections of 25 μm thickness were obtained and stained. Images were obtained using a slide scanner (Aperio digital pathology slide scanner AT2, Leica) at 20× magnification and analyzed using ImageScope (Leica) and ImageJ software. Hemispheric area, hippocampal area including CA1 and CA3 cell body layers were measured from sections corresponding Bregma −1.7 mm to −1.94 mm and Bregma −3.16 mm to −3.4 mm. The densities of CA1 and CA3 cell body layers were measured from middle CA1 and CA3 regions (200 μm in length). Brightness and contrast of grayscale images were adjusted to distinguish cell body layers from adjacent dendritic layers. The areas (pixel numbers) of cell body layers were measured from adjacent three sections for each mouse.

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.

Brain histological sections were prepared using a protocol modified from previous studies of our laboratory (Jeffrey et al. 2014 (link); Stover et al. 2017 (link)). Each mouse was anesthetized by sodium pentobarbital as described above and infused transcardiacally with standard ACSF and then with 10% neutral buffered formalin solution (Sigma-Aldrich). Removed brains were further fixed in a hypertonic formalin solution (with 20% sucrose) for ≥24 h. Brain coronal sections of 50 μm thickness were obtained using a Leica CM3050 research cryostat. Sections were mounted onto glass slides (Superfrost plusmicroscope slides, Fisher Scientific), dried at room temperature for ≥1 week, and then stained with cresyl violet (Sigma Aldrich). Images were obtained using a slide scanner (Aperio digital pathology slide scanner AT2, Leica; at ×20 magnification) and analyzed using ImageScope (Leica) or Image J software (National Institute of Health, USA).