Histological Analysis of Brain Slices

Two central brain slices with the highest radiotracer concentration from autoradiography, as given by autoradiography Conc_mean, per subject were selected for histological analysis. Paraffin embedding, slicing into 5 μm sections and staining for hematoxylin and eosin (H&E) procedures were performed by UAB histology core facility 71 (link). Slices were dewaxed using EZ-DEWAX (HK584, BioGenex, Fremont, CA) twice for 5 minutes, followed by antigen retrieval with citrate buffer using an EZ-Retriever System (MW015-IR, BioGenex, Fremont, CA) and blocking with 5% BSA in TBST for 5 minutes at room temperature. CD8a monoclonal primary antibody (1:100, 4SM15, Invitrogen, Waltham, MA) was incubated overnight at 4 ºC. Anti-Rat-Ig HRP Detection kit (51-7605KC, BD Pharmingen, Franklin Lakes, NJ) was used for the secondary antibody (1:100). DAB substrate (SK-4100, Vector Laboratories, Newark, CA) was incubated for 7 minutes at room temperature for the development of the stain. Scanning of H&E and IHC stained slides was performed using an EVOS M7000 Imaging System (Thermo Fisher, Waltham, MA) at 20x magnification.