Histological Analysis of Hippocampal Damage

Rats were anesthetized with 4% choral hydrate (10 ml/kg) and then perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and post-fixed for 24 hours in the same fixative. Post-fixed brains were embedded in paraffin and sliced on a microtome at a 5 μm thickness. The sections between 2 mm and 3 mm posterior of the bregma were used for this study. For histological assessment of damage to the hippocampus, the paraffin-embedded brain sections were mounted in Poly-L-Lysine-coated slides for histopathological examination by H&E staining. The sections were also incubated in 1% cresyl violet for Nissle staining. To determine CHOP and p-JNK activities, sections were incubated with 0.3% H₂O₂ in methanol for 30 minutes, and then submerged into blocking solution (1% albumin bovine in PBS) for 1 hour at room temperature prior staining. Primary antibodies, such as CHOP (1:200) and p-JNK (1:500) were applied on sections with overnight incubation at 4°C. After 3 times of PBS wash, they were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 hours at 37°C. The reaction was terminated with 3, 3′-diaminobenzidine, and images were captured at 400x magnification using a Nikon ECLPSE 80i. The optical density of CHOP and p-JNK within the injury site in hippocampus were counted at five randomly selected fields per sample [55 (link)].