Neutral buffered formalin solution

Following the experiment, all mice were anesthetized in a chamber using 2% isoflurane (Virbac, UK). Brain tissues were extracted for histopathology analysis and fixed with 10% neutral buffered formalin solution (BBC Biochemical, Mount Vernon, WA, USA) for 1 week. The fixed tissues were embedded in a paraffin block and sectioned into 4-μm thick sections. The sections were then mounted on slides and stained with hematoxylin and eosin (H&E) solution using an autostainer (Dako Coverstainner; Agilent, Santa Clara, CA, USA). Additionally, the tissue sections were stained immunohistochemically using the labeled polymer DAKO EnVisionTM+System-HRP (Agilent) according to the manufacturer’s instructions. The brain sections were stained with anti-GFAP (ab7260; Abcam, Cambridge, MA, USA), anti-Iba1 (ab5076; Abcam, Cambridge, MA, USA), and anti-NeuN (ab177487; Abcam, Cambridge, MA, USA) primary antibody. After staining, all areas of the brain that were scanned with a slide scanner (Pannoramic SCAN II; 3DHISTECH, Budapest, Hungary) and were captured by a slide viewer (CaseViewer; 3DHISTECH, Budapest, Hungary). For quantification analysis, Each slide captures 3 points at 400x in the hippocampus. Using image analyzer software Image J (NIH, MD, USA), the positive portion of the total is quantified as a percentage and the average is calculated.