Avidin biotin peroxidase complex

Mouse brains were prepared for immunolabeling EM. Briefly, 4-week-old mice were transcardially perfused with 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde in 0.1 M PB for 30 min. Brains were removed after perfusion and post-fixed for 12 h at 4 °C. Coronal sections (100 μm thick) were sectioned using a vibratome (Leica VT1000 S).
For post-embedding immunogold labeling, brain sections crossing the cerebellum or cerebral cortex were post-fixed in 0.5% osmium tetroxide and processed for Epon 812 embedding. The ultrathin sections were stained with specific rabbit anti-PRRT2 antibodies (1:200; #3) and then goat anti-Rabbit IgG with 15 nm immunogold (1:100; #25113, EMS, USA).
For pre-embedding immunoperoxidase staining, vibratome sections of mouse cerebellum and cerebral cortex segments were immunostained with our rabbit anti-PRRT2 antibodies (1:200) followed by biotinylated goat anti-rabbit IgG (1:200) and then avidin-biotin-peroxidase complex (1:100; Sigma). The sections were incubated in 0.01 M PBS containing 3, 3′-diaminobenzidine and hydrogen peroxide. They were then post-fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated and processed for Epon 812 embedding. To distinguish the vesicles with PRRT2-immunoreactivity from unlabeled vesicles, we did not counterstain the ultrathin sections with uranyl acetate and lead citrate.

Immunohistochemistry was performed to detect the BBB impairment during the acute phase after TBI. Briefly, after anesthetization with 3% pentobarbital sodium (100 mg/kg), the brains of rats were removed and stored in 4% paraformaldehyde until processing. Coronal sections were cut on a cryostat at 10 μm thickness, deparaffinized and rehydrated. Sections were permeabilized with 3% H₂O₂ for 10 min and blocked with 5% normal donkey serum in PBS for 60 min at room temperature. Immunostaining was performed by incubating the sections with primary antibodies (antibodies are listed in Table 1) against ZO-1, MMP-9 and gp91^phox at 4 °C overnight, followed by staining with biotin-labeled secondary antibodies for 120 min and incubation with an avidin-biotin-peroxidase complex (1:100, Sigma, USA) for 1 h at 37 °C. Immunoreactivity was visualized with diaminobenzidine (Boster Biotech Co. Wuhan, China).
ZO-1, MMP-9 and gp91^phox positive staining (brown yellow) was identified under a light microscope. For the image analysis. Ten microscopic fields were randomly selected randomly from each group, imaged at of 400× magnification, and the integral optical density (IOD) for each group was automatically measured using the digital software Image ProPlus 5.0 (Media Cybernetics, USA).