Quantifying Tau Pathology in Alzheimer's Disease

Tissue was fixed in periodate-lysine-paraformaldehyde, tissue blocks were paraffin-embedded, and sections were cut at 10μm for immunohistochemistry. Antigen retrieval for and Aβ was performed with formic acid treatment for two minutes. Sections were incubated overnight at 4°C with antibodies to phosphorylated PHF-tau (AT8; Pierce Endogen, Rockford IL; 1:2000) and Aβ (4G8; BioLegend, San Diego, CA; 1:100,000). Sections were washed three times with PBS (pH 7.4), and subsequently treated with biotinylated secondary antibody and labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories). The sections were then counterstained with Gill’s Hematoxylin (Vector Laboratories H-3401) and coverslipped using Permount mounting medium. For tau pathology quantification, slides immunostained for ptau (AT8) were scanned at 20× magnification with a Leica Aperio Scanscope (Leica Biosystems, Richmond, IL) as previously described [13 (link)]. ImageScope (Leica Biosystems) was used to highlight the gray matter at the bottom third of the sulcus. Leica’s image analysis and automated counting software (Aperio positive pixel count, Version 9) was calibrated for staining intensity to detect AT8-immunoreactivity within the region of interest. Counts were normalized to the area measured and are presented as positive pixels per mm² within the sulcal depth.