Immunostaining of Brain Tissue

For immunostaining, the brain tissue was pretreated with incubation in 1.2% hydrogen peroxide for 15 min followed by washing in 0.01% PBS. Tissues were stained with several primary antibodies diluted in PBS containing 0.3% Triton X-100. The primary antibodies used in this study are as follows: PKM2 (diluted 1:500, Cell Signaling Technologies, Danvers, MA, USA); S100B (diluted 1:500, Synaptic Systems, Goettingen, Germany); 4-HNE (diluted 1:500, Alpha Diagnostic Intl. Inc., San Antonio, TX, USA); MAP2 (diluted 1:200, Alpha Diagnostic Intl. Inc.); LDHA (diluted 1:100, Sigma-Aldrich Co., St. Louis, MO, USA); LDHB (diluted 1:100, Sigma-Aldrich Co.); NeuN (diluted 1:500, Billerica, Millipore Co., Burlington, MA, USA); MCT4 (1:150, Santa Cruz Biotechnology, Dallas, CA, USA); MCT2 (1:1000, Invitrogen, Waltham, MA, USA). Then, we washed the brain tissue with 0.01 M PBS and stained with the appropriate secondary antibody (Alexa-Fluor-594-conjugated IgG secondary antibody and Alexa-Fluor-488-conjugated secondary antibody, both diluted 1:250, Invitrogen, Grand Island, NY, USA). To confirm staining, brain tissue was examined under a microscope using gelatin-coated slides. The ImageJ program (NIH, Bethesda, Rockville, MD, USA) was used to analysis the intensity [3 (link),31 (link)].