Immunofluorescence

The presence and localization of tau protein in oral mucosa cells was analyzed by confocal microscopy. Two forms of phosphorylated Tau were examined, one epitope associated with normal and pathological Tau phosphorylation (17, 32) monoclonal rabbit anti p-Tau (Ser396) (Abcam, Cambridge, MA, USA, dilution 1:100) and a second epitope that characterizes Tau hyperphosphorylation monoclonal mouse anti p-Tau (Ser202 + Thr205, Thermo Fisher Scientific, Rockford, IL, USA dilution 1:100). Nuclear localization was explored employing monoclonal rabbit anti-Lamin-A (Abcam, Cambridge, MA, USA, dilution 1:100). Antibodies were incubated overnight at 4°C and then rinsed. For detection, goat α-rabbit Cy5 (Life Technologies, USA, dilution 1:200) and goat α-mouse Alexa Fluor 488 (Thermo Fisher Scientific, Rockford, IL, USA, dilution 1:300) were incubated for 2 h at room temperature, followed by SYTOX (Molecular Probes, Eugene, OR, USA, dilution 1:1,000) for 30 s, then rinsed and mounted with Vectashield^® (Vector Laboratories, Burlingame, CA, USA). Analysis was performed with a LEICA TCS SP2 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany).