Application system modules for histological analysis

Six random non-overlapping fields were scanned and analyzed for determining the positive mean area percentage of immuno-histochemical expression levels of GFAP and Beta amyloid as well as mean numbers of IbA-1/++ microglial cells in each immuno-stained tissue section CA3 subregions. All morphological examinations and quantitative analysis were recorded using Leica Application system modules for histological analysis (Leica Microsystems GmbH, Wetzlar, Germany).

According to the manufacturer's protocols and instructions, immunohistochemical staining for Iba1 and glial fibrillary acidic protein (GFAP) was performed. Antigen retrieved brain tissue sections were blocked by 3% hydrogen peroxide for 15 min then they were incubated with the primary antibodies; anti-GFAP (Cat. No. 13-0300; Thermo Scientific Co., MA, USA; 1:100) and anti-Iba1 antibodies (Cat. No. ab108539; Abcam, MA, USA; 1:100) overnight at 4°C. Afterwards, they were washed with phosphate-buffered saline then incubated for 20 min with biotinylated link antibody and peroxidase-labeled streptavidin (Dako, Carpinteria, CA, USA). The reaction was visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB Substrate Kit, Vector Laboratories Inc., Burlingame, CA, USA). Sections were counterstained with hematoxylin, dehydrated, and cleared in xylene then examined via light microscope.
For immunohistochemical quantitative analysis, six random striatal non overlapping fields were scanned and analyzed for determining the positive mean area percentage of GFAP immunoexpression and the mean number of Iba1-positive microglial cells in each immunostained tissue section. All morphological examinations, photographs as well as quantitative analysis were recorded using Leica Application system modules for histological analysis (Leica Microsystems GmbH, Wetzlar, Germany).

We analyzed six random non-overlapping fields to determine the positive mean area percentage of immunohistochemical expression levels of GFAP and Aβ in each immune-stained tissue section. We also counted the IbA-1/++ microglial cells in six random non-overlapping fields in each immune-stained tissue section. We performed all morphological examinations, imaging, and quantitative analysis using Leica Application system modules for histological analysis (Leica Microsystems GmbH, Wetzlar, Germany).