Quantifying 5-HT2A Receptors in TBI

5-HT_2A binding was analyzed in membrane preparations as previously described (Canal et al., 2010 (link)). Three or 10 days following mTBI or sham treatments, subjects were sacrificed via rapid decapitation and bilateral frontal cortex samples were dissected on ice. Samples were homogenized in 3 ml ice-cold Tris binding assay buffer (50 mM Tris-HCl, 10 mM MgCl₂, 0.1 mM EDTA, pH = 7.4), and subsequently centrifuged at 20,000 x g for 20 min at 4°C. The supernatant was decanted, and the pellet was resuspended in a 1.5-ml binding assay buffer. Protein concentrations were measured with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Membrane preparations (200 µg protein) were incubated with the 5-HT_2A antagonist [³H]ketanserin (PerkinElmer, Waltham, MA) (1 or 10 nM) at 37°C for 60 min. Samples were run in duplicate and nonspecific binding was determined in the presence of 100 µM methysergide (Sigma Aldrich, St Louis, MO). Samples were collected using a Brandel cell harvester and washed with ice-cold phosphate-buffered saline (PBS, pH = 7.4). Samples were incubated in 7 ml scintillation fluid overnight (National Diagnostics) and radioactive counts were measured via scintillation spectrometry (Beckman Coulter).