*4.10. Evaluation of Hippocampal Astrocytes and Microglia*

To analyze astrocyte and microglial activation, we performed Iba-1 and GFAP staining. Staining was used with a mixture of goat antibody to mouse Iba-1 (diluted 1:500, Abcam, Cambridge, UK) and rabbit antibody to mouse GFAP (diluted 1:1000, Abcam, Cambridge, UK). Following incubation in 0.01 M PBS containing 0.3% TritonX-100, we left it overnight in a 4 ◦C incubator. After overnight incubation, we washed the sections three times for 10 min with 0.01 M PBS. Then, the sections were soaked in a secondary antibody (Alexa-Fluor-488-conjugated donkey anti-rabbit IgG secondary antibody and Alexa-Fluor-594-conjugated donkey anti-goat IgG secondary antibody, both diluted 1:250, Invitrogen, Grand Island, NY, USA) for 2 h at RT. The brain sections were raised on gelatin-coated slides for analysis under a microscope. We used the Image J (NIH, Bethesda, Rockville, MD, USA) program to measure the astrocytes. In the case of microglia, five brain sections were scored with the same area (20× magnification) of the hippocampal CA1 region. The functional standards of microglia cells were their number, morphology, and intensity of microglia activation. Iba-1-immunoreactive cell score of 0: no cells are present; 1:1–9 cells; 2:10–20 cells; and 3:>20 cells with continuous processers per 100 μm2. Morphology score of 0:no activated morphology (amoeboid morphology with enlarged soma and thickened processes); 1:1–45% of microglia activation; 2:45–90% of microglia activation; and 3: >90% of microglia with the activated morphology. The intensity of microglial activation was measured using the Image J (NIH, Bethesda, Rockville, MD, USA) program. After the measurements, an intensity score of 1:0–19% expression; 2:20–29% expression; and 3:>29% expression. Therefore, the total score summed up the three scores depending on the categories, ranging from 0 to 9 [18,81,82].
