AT8 immunohistochemistry

Study mice were perfused with PBS (20 ml) after being deeply anesthetized using a protocol approved by the University of Pennsylvania IACUC. The brains were subsequently removed and one hemisphere from each mouse was processed as previously described [25, 27], with 6 μm thick paraffin-embedded sections prepared and stained with the AT8 antibody (1:2000 dilution) that recognizes tau phosphorylated at S202/T205 [45]. Immunostained sections that were masked to treatment group were imaged using a 4× microscopic objective. For analysis of hippocampal neurons, 3 matched brain sections (Bregma: − 2.20 to − 2.80) from vehicle- and 51657-treated PS19 mice were manually annotated around the entire hippocampus and entorhinal cortex using HALO (Indica Labs, Corrales, NM) software. Sections representing average AT8 staining intensity were thresholded to allow quantification of tau pathology in the hippocampal and cortical sections without contribution of background staining, and a common threshold was then applied to all sections. Quantification was conducted with the HALO software. The area of tau pathology within each annotated region was determined, and this was summed across the three individual sections from each mouse and divided by the sum of the total annotated area from the three sections to get the total % area with tau pathology. This value was multiplied by the average optical density (OD) of the tau pathology to yield the final “normalized AT8 area x OD”, and the sum of these values from the hippocampal and cortical assessments are reported.