Radiolabeling and Quantification of TSPO in Cardiac Tissue

[¹²⁵I]-IodoDPA-713, used to assess TSPO levels in the heart, was synthesized using a two-step reaction process, as previously [29 (link)]. Desmethyl DPA-713 was first iodinated with [¹²⁵I]NaI in the presence of iodogen. The ¹²⁵I radiolabeled intermediate was methylated by MeI/K₂CO₃/DMF and HPLC purified. The overall radioactive yield was 35-50% with >99% radiochemical purity. Fresh-frozen hearts were sectioned (20μm) on a cryostat in the sagittal plane and thaw-mounted onto poly-L-lysine-coated slides and stored at −20°C. Slides were dried for 30min and prewashed with 50mM Tris-HCl buffer (pH 7.4). To determine total binding, slides were incubated for 1h at RT in buffer containing 0.8nM [¹²⁵I]-IodoDPA-713 (specific activity 2200 Ci/mmol). To measure non-specific binding, adjacent slides were incubated in the presence of 50μM cold racemic PK11195 (Sigma, St. Louis, MO). After incubation, slides were washed and exposed to film at RT for 20min. Images were captured and analyzed using MCID (InterFocus Imaging Ltd., Cambridge, England) calibrating for density using [¹²⁵I]-microscales. The mean value for all sections/heart was used for statistical analyses.