*4.11. Evaluation of BBB Disruption*

To analyze the putative breakdown of the BBB, we used immunohistochemistry to find serum IgG leakage [83]. To detect IgG-like immunoreactivity, the ABC immunoperoxidase protocol was used [84]. Mouse brains were fixed by transcardiac perfusion with 0.9% normal saline, followed by 4% paraformaldehyde. We used anti-mouse IgG (diluted 1:250, Burlingame, Vector, CA, USA) which can discover leakages of IgG when the BBB is damaged. After washing in 0.01 M PBS, brain sections were deeply soaked in the ABC complex mixture (Vector, Burlingame, CA, USA) for 2 h at RT. The immunoreactivity was visualized with 0.06% 3,3'-diaminobenzidine (DAB ager, Sigma–Aldrich Co., St. Louis, MO, USA) in 0.1 M PBS buffer. Leaked IgG extravasations were detected using a bright-field microscope.

#### *4.12. Evaluation of Live Hippocampal Neurons*

To assess the number of live neurons present in a sample, NeuN was detected by immunofluorescence staining. NeuN antibodies (diluted 1:500, EMD Millipore, Billerica, MA, USA) for immunohistochemical staining were used as in previous studies. Brain sections were soaked in a monoclonal rabbit anti-NeuN serum with PBS containing 0.3% TritonX-100 overnight in a 4 ◦C

incubator. After overnight incubation, brain sections were washed three times for 10 min each with 0.01 M PBS, and then the brain sections were also soaked in a solution of Alexa-Fluor-594-conjugated donkey anti-rabbit IgG secondary antibody (diluted 1:250, Invitrogen, Grand Island, NY, USA) for 2 h at room temperature (RT). The brain sections were raised on gelatin-coated slides for analysis under a microscope. A blinded observer counted the NeuN-positive cells. NeuN-positive cells were counted and evaluated in the hippocampal Sub, CA1, CA2, and CA3 regions from each hemisphere. The total number of NeuN-positive cells from the hippocampal region was used for statistical analysis [18].
