Quantifying Inflammatory Markers in Brain Tissue

COX-2, p-NF-κB, and TNF-α expression were quantified using rat ELISA kits following the manufacturer's instructions. Briefly, an appropriate quantity of brain tissue (50 mg) was homogenized using a Heidolph crusher at 15,000 rpm in 2500 μL PBS containing PMSF as the protease inhibitor [56 (link)]. Next, the tissue homogenate was centrifuged at 4000×g for 10 min, and the supernatant was collected. Total protein concentration in the supernatant of each group was calculated using the BCA method (Elabscience); moreover, an equivalent protein quantity was used to quantify the protein concentration of COX-2, p-NF-κB, and TNF-α using an ELISA microplate reader (BioTek EL×808). Finally, the protein concentration (pg/mL) was normalized to the total protein content (pg/mg total protein).