Rabbit anti hmgb1

Rats were deeply anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA) at desired time points post-eFSE. Brains were removed and post-fixed in 4% PFA for 90 min. Brains were then cryoprotected in 30% sucrose, rapidly frozen, and stored at −80°C. Thirty micrometer sections of dorsal hippocampus were obtained on a cryostat and stored in antifreeze at 4°C until use. Serial sections were blocked in 10% normal goat serum and 0.03% Triton X in 1× PBS for 1 h at 4°C. Primary antibodies were incubated in 4% normal goat serum with 0.03% Triton X overnight at 4°C. The following antibodies were used: rabbit anti-HMGB1 1:1000 (Abcam), mouse anti-GFAP 1:3000 (Millipore), and mouse anti-IBA1 1:4000 (Wako). Sections were washed with 1× PBS, and the reaction product was visualized using 3,3'-diaminobezidine. Colocalization of cell markers with HMGB1 was achieved by coincubating rabbit anti-HMGB1 1:1000 (Abcam) with the following antibodies: mouse anti-NeuN (Chemicon), mouse anti-GFAP 1:3000 (Millipore), and mouse anti-CD11b (ABD Serotec). After 24 h of incubation, sections were washed in 1× PBS and then incubated in the appropriate secondary antibodies conjugated with Alexa Fluor 568 or Alexa Fluor 488. Colocalization was visualized using confocal microscopy.

HMGB1 protein levels in LC brain homogenates were determined by western blot analysis, and normalized to a GAPDH loading control. 20μg of each homogenate was separated by SDS-PAGE, transferred to a PVDF membrane, and blocked using Blok fluorescent blocker (Millipore, Billerica, MA). The PVDF membranes were incubated with rabbit anti-HMGB1 (1:5000) and mouse anti-GAPDH (1:1000; Abcam, Cambridge, MA) overnight at 4°C. The secondary antibodies goat anti-rabbit Dylight 680 and goat anti-mouse Dylight 800 (Thermo Fisher, Waltham, MA) were diluted 1:5000. HMGB1 and GAPDH bands were quantitated using LI-COR Odyssey Image Studio software (LI-COR, Lincoln, NE).