*4.9. Evaluation of Hippocampal Microtubule Damage*

To analyze microtubule damage from the brain sections, MAP2 was detected by immunofluorescence staining. MAP2 antibodies (Alpha Diagnostic Intl. Inc., San Antonio, TX, USA) for immunohistochemical staining were used as in a previous study [80]. Brain sections were soaked in a polyclonal rabbit anti-MAP2 serum (diluted 1:200, Alpha Diagnostic Intl. Inc., San Antonio, TX, USA) with PBS containing 0.3% TritonX-100 overnight in a 4 ◦C incubator. After overnight incubation, we washed the sections three times for 10 min with 0.01 M PBS, and then the brain sections were soaked in a solution of Alexa-Fluor-488-conjugated donkey anti-rabbit IgG secondary antibody (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 microtubule damage and measured the mean gray value.
