2.9. Western Blot

To detect BDNF and TrkB protein expression, total proteins were extracted from the brain tissue in RIPA lysis buffer. The protein concentration was determined using the BCA kit. Sixty μg of the proteins was separated by SDS-PAGE and transferred (Bio-Rad, USA) to PVDF membranes (Millipore, USA). BDNF and TrkB protein expression levels were quantified using rabbit polyclonal antibodies specific for each protein. The expression levels of these proteins were standardized to human GADPH using a mouse polyclonal anti-GADPH antibody. Primary antibodies were detected using goat anti-rabbit or goat anti-mouse horseradish peroxidase- (HRP-) conjugated secondary antibodies. Immunoreactive bands were visualized using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer, USA) according to the manufacturer's instructions and then quantified by densitometry using a Bio-Rad gel imaging system and Quantity One software.