Postmortem brain tissue was fixed in periodate-lysine-paraformaldehyde (PLP) for at least 3 months at 4 °C. The neuropathological assessment was performed using procedures previously established [58 (link), 76 (link)]. Neuropathological evaluations were made by board-certified neuropathologists (ACM, TDS, BRH) according to published diagnostic criteria and were kept blinded to antemortem clinical information [58 (link)]. Cerebral arteriolosclerosis, atherosclerosis, and CAA were evaluated on a semiquantitative scale [12 (link), 73 (link)].
For immunohistochemical assessment, 20 µm slides from paraffin-embedded tissue blocks from the DLF were prepared and stained for AT8 as previously described [16 (link)]. Slides were scanned, digitized at 20× magnification, and analyzed for AT8 density (total area in the sulcus positive for AT8 staining divided by the total area of tissue analyzed) using Aperio Scanscope (Leica) as previously described [17 ]. The depth of the cortical sulcus was defined as the bottom third of two connecting gyri. Ki67 staining was completed using Leica’s Ready-to-Use Ki67 antibody reagent on the BOND staining system (Leica, Deer Park, IL) and imaged on the Vectra Polaris slide scanner (Akoya, Marlborough, MA).
For immunohistochemical assessment, 20 µm slides from paraffin-embedded tissue blocks from the DLF were prepared and stained for AT8 as previously described [16 (link)]. Slides were scanned, digitized at 20× magnification, and analyzed for AT8 density (total area in the sulcus positive for AT8 staining divided by the total area of tissue analyzed) using Aperio Scanscope (Leica) as previously described [17 ]. The depth of the cortical sulcus was defined as the bottom third of two connecting gyri. Ki67 staining was completed using Leica’s Ready-to-Use Ki67 antibody reagent on the BOND staining system (Leica, Deer Park, IL) and imaged on the Vectra Polaris slide scanner (Akoya, Marlborough, MA).