Kosmidis S, Grammenoudi S, Papanikolopoulou K, Skoulakis EM. Differential effects of Tau on the integrity and function of neurons essential for learning in Drosophila. J Neurosci. 2010 Jan 13;30(2):464-77. PubMed Abstract

REAGENTS/MATERIAL:
Immunohistochemistry on paraffin sections was performed essentially as described previously. Rabbit anti-LEO/Leonardo (Skoulakis and Davis, 1996) was used at 1:4000 and rabbit anti-DRK (Moressis A, Friedrich AR, et al, 2009) at 1:1500. Sections from all strains were obtained and processed in parallel in each experiment and were evaluated for MB morphology without knowledge of the genotype. The anti-ELAV (Developmental Studies Hybridoma Bank) was used at 1:200 and anti-DAC was used at 1:8.
For Western blotting, Drosophila tissue (adult heads, embryos, or larvae) were homogenized in 1x Laemli's buffer, and the extracts were heated for 10 min at 95°C, centrifuged at 8000 x g for 5 min, and separated in SDS-acrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes and probed with mouse monoclonal anti-Tau 46 (Zymed Laboratories), which targets the C terminus of the protein at 1:3000, and mouse monoclonal TAU5 (Calbiochem), which targets the proline-rich domain (PRD), mouse monoclonal anti-tau AT100 (Pierce Endogen) at 1:250, the polyclonal rabbit antibodies anti-tau pS262, anti-tau pS356, and anti-Paired Helical Filament (PHF) (Biosource), and monoclonal antibody AT8 (kindly provided by A. Mudher) was used at 1:200. To normalize for sample loading, the membranes were concurrently probed with mouse monoclonal anti-syntaxin (8C3) (Developmental Studies Hybridoma Bank). Proteins were visualized with chemiluminescence.
We thank A. Mudher (University of Southampton, Southampton, UK), and the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA) for antibodies.