Characterizing Tau Protein in Drosophila Brains

Fly heads (2 μL/per head, 30 heads/blot) were homogenized in cold Tris buffer (25 mM Tris-HCl, pH 7.4, 15 mM NaCl, 1 mM EGTA, 1 mM ethylenediaminetetraacetic acid, supplemented with protease and phosphatase inhibitors) and centrifuged at 3000 × g for 3 minutes, followed by a 1 hour spin of the supernatant at 100,000 × g. Sarkosyl extraction, SDS-PAGE, and western blotting were carried out as described (Colodner and Feany, 2010 (link)). The primary anti-Tau antibodies were HT7 (1:500), Tau46 (1:500), AT270 (1:5000), AT8 (1:100), and AT180 (1:500). HT7 and Tau46 are phosphorylation-independent and specific for the amino- and carboxy-terminal regions of Tau, respectively. AT270, AT8, and AT180 are phosphorylation-dependent and recognize pT181 (AT270), pS202, pT205, and pS208 (AT8) (Malia et al., 2016 (link)), and pT231 (AT180) in Tau. The anti-beta actin antibody was used at 1:1000. Except for anti-beta actin (Abcam), all antibodies were from Thermo Scientific Pierce. For immunohistochemistry, paraffin-embedded brain sections (4 μm) were incubated with primary antibody (HT7, 1:400; AT8, 1:400, MC-1, 1:100) for 24 h at 4 °C, washed, incubated with secondary antibody, and the signal visualized using a Vector VIP substrate kit (Vector Laboratories) with an Olympus BX41 microscope equipped with an integrated 3.0 megapixel CMOS camera.