*4.8. Evaluation of Hippocampal Oxidative Stress*

To analyze oxidative damage induced by the lipid peroxidation product from the brain sections, 4HNE was detected by immunofluorescence staining. 4HNE antibodies (Alpha Diagnostic Intl. Inc., San Antonio, TX, USA) for immunohistochemical staining were used as in previous studies [77,78]. Brain sections were soaked in a monoclonal mouse anti-4HNE serum (diluted 1:500, Alpha Diagnostic Intl. Inc., San Antonio, TX, USA) with the PBS containing 0.3% TritonX-100 overnight in a 4 ◦C incubator. After overnight incubation, brain sections were washed three times for 10 min with 0.01 M PBS, and then the brain sections were also soaked in a solution of Alexa-Fluor-594-conjugated donkey anti-mouse 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. We used the Image J (NIH, Bethesda, Rockville, MD, USA) program to measure the oxidative injury and measured the mean gray value [79].
