Ab6013

The rats were anesthetized and decapitated at 6, 12, or 24 h after TBI and perfused through the heart with 4% paraformaldehyde. Next, the mPFC and LHA were carefully separated and fixed in 10% formaldehyde, dehydrated in graded ethanol, paraffin-embedded, and sectioned at 4 μm coronal thickness for examination. Slides were baked at 65°C for 1 h after preheating followed by deparaffinization with xylene and rehydration. The slices were then placed in citrate buffer solution at high temperature for 20 min and then naturally cooled to room temperature. Afterward, the sections were treated with 0.3% hydrogen peroxide for 10 min, rinsed three times for 5 min each, and incubated with normal goat serum for 30 min. The sections were then incubated overnight at 4°C with rabbit anti-OX1R (1 : 250, Ab68718; Abcam) antibody and orexin antibody (1 : 250, Ab55051; Abcam). Following incubation, the tissue sections were rinsed extensively with PBS and then incubated with a biotinylated goat anti-rabbit antibody (1 : 2000, Ab6013; Abcam). Finally, the sections were reacted with diaminobenzidine and visualized under a florescence microscope (Nikon, Tokyo, Japan).

Cells collected after 72 h treatments were lysed in RIPA buffer (Millipore) containing protease inhibitor cocktail (cOmplete, Roche). Protein concentrations were measured colorimetrically using Pierce BCA protein assay kit (Thermo Scientific). Total protein concentrations of 15 µg for cyclin D1 and 40 µg for p21 in loading buffer (Roti-Load 1, Carl Roth) were separated by SDS-PAGE in a 12% separating gel and 5% stacking gel (Rotiphorese NF-Acrylamide/Bis-solution 30% (29:1), Carl Roth). Proteins were transferred to PVDF membrane (Immobilon P, Millipore) followed by blocking (Roti-Block, Carl Roth), incubation in primary antibody and then secondary antibody. Rabbit monoclonal to p21 [EPR3993] (1:1000, ab109199, Abcam), Rabbit monoclonal to cyclin D1 [EPR2241] (1:3000, ab134175, Abcam) and Rabbit polyclonal to GAPDH (1:1000, ab9485, Abcam) were used as primary antibodies. The secondary antibody used was HRP conjugated Goat F(ab’)2 Anti-Rabbit IgG (1:7500, ab6013, Abcam). The membrane was washed between incubations with TBST (20 mM Tris–HCl (pH 7.6), 0.137 M NaCl, 0.05% Tween 20). The blots were then developed using Pierce ECL plus (Thermo Scientific) and imaged using Fusion FX (Vilber). The blots were analyzed densitometrically using ImageJ (https://imagej.nih.gov).