Application module

The hippocampal proliferative activity was measured using Ki-67 immunohistochemistry to elucidate β-catenin's compensatory mechanism [52] (link). Tissue sections with a thickness of 5 μ were produced in paraffin, and the manufacturer's protocol for immunohistochemistry was followed. Deparaffinized recovered tissue slices were treated with 0.3% H 2 O 2 for 20 min. Then, at 4 °C overnight, brain samples were treated with Anti-ki67 (Cat. No. GTX16667, Genetex Co., USA, 1:100). Tissue sections were rinsed in phosphate buffered saline and treated with the secondary antibody HRP Envision kit (DAKO, USA) for 20 min before being washed and incubated with diaminobenzidine for 15 min. After that, tissue sections were washed by phosphate-buffered saline, and then hematoxylin counterstained, dehydrated, and cleared in xylene before being cover slid for microscopic analysis. Three random non-overlapping fields were selected and scanned from dentate gyrus (DG) regions of each sample for the determination of area-based percentage of immunoexpression levels of Ki-67 in immunohistochemically stained sections. All light microscopic examination and data were obtained using Leica Application module for histological analysis attached to full HD microscopic imaging system (Leica Microsystems GmbH, Germany) [53] (link).

Brain (cerebral cortex) tissue samples (n = 3 per each group) were fixed in 10% neutral buffered formalin for 72 h, then processed in serial grades of alcohol, cleared in xylene, infiltrated, and embedded into Paraplast tissue embedding media. Then, 5-µm thick sagittal sections were cut by a rotatory microtome for demonstration of the whole cerebral cortex and mounted on glass slides. Tissue sections were stained by hematoxylin and eosin (H&E) as a general morphological examination staining method, then examined by the Leica Application module attached to the full HD microscopic imaging system (Leica Microsystems GmbH, Germany). All standard procedures were done according to Culling [45] .