Human frontal cortex sections were deparaffinized followed by antigen retrieval as described above. Sections were permeabilized in 0.2% Triton X-100 in PBS, blocked with 2% goat serum in PBS and incubated overnight at 4 °C with primary antibodies to pSRRM2 (mouse mAb clone SC-35, [24] and pS422 (rabbit mAb clone EPR2866) or DAKO anti-tau (rabbit polyclonal anti-tau). For double label immunofluorescence, secondary antibodies AlexaFluor 488 goat anti-rabbit and AlexaFluor 647 goat anti-mouse secondary antibodies (ThermoFisher, Waltham, MA) were applied for 45 min at room temperature and sections were counterstained with 300 nM DAPI before mounting with ProLong Gold Antifade. Images were obtained on a DeltaVision Elite microscope (Cytava Lifesciences, Marlborough, MA) using a 100X oil immersion objective and colocalization analysis was performed with SoftwoRx 6.0 Beta software (Cytiva).
