The rats were anesthetized and decapitated at 6, 12 and 24 hours after TBI, and perfused through the heart with 4% paraformaldehyde. Next, the brains were carefully removed, and 40-µm-thick coronal sections were cut for examination. The sections were rinsed with phosphate-buffered saline and treated with 0.3% hydrogen peroxide (H2O2) for 30 minutes. Afterwards, the sections were rinsed three times for 5 minutes each and then incubated with normal goat serum for 20 minutes. The sections were then incubated overnight at 4°C with rabbit anti-OX1R antibody (1:200; ab68718, Abcam). Following incubation, the tissue sections were extensively rinsed with phosphate-buffered saline and then incubated with a biotinylated goat anti-rabbit antibody. Finally, the sections were reacted with diaminobenzidine and visualized under a light microscope (BX511T-PHD-J11, Olympus, Tokyo, Japan).
Protein expression was quantified as a function of the percentage and staining intensity of immunoreactive cells (Soslow et al., 2000) as follows: A × B, where A represents the percentage of positive cells (0–4, where 0 = 0–1%, 1 = 1–10%, 2 = 10–50%, 3 = 50–80%, 4 = 80–100%) and B represents the intensity of staining (0–3, where 0 = no significant staining, 1 = mild staining, 2 = moderate staining, 3 = dark staining).
Protein expression was quantified as a function of the percentage and staining intensity of immunoreactive cells (Soslow et al., 2000) as follows: A × B, where A represents the percentage of positive cells (0–4, where 0 = 0–1%, 1 = 1–10%, 2 = 10–50%, 3 = 50–80%, 4 = 80–100%) and B represents the intensity of staining (0–3, where 0 = no significant staining, 1 = mild staining, 2 = moderate staining, 3 = dark staining).