Anti ptauser202 thr205 at8

For protein extraction in flies, 10 heads of each group were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, R0278). Samples were mixed with Laemmli sample buffer containing 2% SDS, 10% glycerol, 62.5 mM Tris-HCl (pH 6.8), 0.001% bromophenol blue, and 5% β-mercaptoethanol, and heated at 95 °C for 10 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking at room temperature for 1.5 hr, the membrane was incubated with primary antibody at 4 °C overnight, followed by secondary antibody for 1.5 hr at room temperature. Imaging was performed on an Odyssey Infrared Imaging system (LI-COR Biosciences) and analyzed using Image Studio (ver 4.0). Primary antibody dilutions were used as follows: anti-pTau^{Ser202/Thr205} (AT8, 1:500; ThermoScientific, Rockford, IL, USA), anti-pTau^Ser262 (1:1000; ThermoScientific), anti-β-actin (1:5000, Sigma-Aldrich, St Louis, MO, USA), anti-total-Tau (5A6, 1:250, Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Fly brains with attached lamina were dissected as previously described (Brazill et al., 2018 (link)). Salivary glands were dissected from wandering third-instar larvae (L3). Samples were fixed in freshly made 4% formaldehyde for 15 min, washed in PBS containing 0.4% (v/v) Triton X-100 (PBTX), and incubated with primary antibodies at 4°C overnight. Samples were then washed with PBTX and incubated with secondary antibodies at room temperature for 2 hr. After that, samples were stained with 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Carlsbad, CA, USA) for 10 min and mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories Inc, Burlingame, CA, USA). Samples were kept at 4°C until imaging. The following antibodies were used in this study: anti-Brp (1:250, Developmental Studies Hybridoma Bank, East Iowa City, IA, USA), anti-pTau^Ser262 (1:250, Santa Cruz Biotechnology, CA, USA), anti-pTau^{Ser202/Thr205} (AT8, 1:250, Thermo Fisher Scientific, Carlsbad, CA, USA), and anti-Drosophila Nmnat (1:500; Zhai et al., 2006 (link)).