Immunohistochemistry of Drosophila Brain and Salivary Gland

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)).