Immunostaining of Neuron Cytoskeleton

Primary fly or frog neurons were fixed in 4% paraformaldehyde (PFA; in 0.05 M phosphate buffer, pH 7–7.2) for 30 min at room temperature (RT). For anti-Eb1 and anti-GTP-tubulin staining, cells were fixed for 10 mins at -20°C in +TIP fix (90% methanol, 3% formaldehyde, 5 mM sodium carbonate, pH 9; stored at -80°C and added to the cells [49 (link)]), then washed in PBT (PBS with 0.3% TritonX). Antibody staining and washes were performed with PBT. Staining reagents: anti-tubulin (clone DM1A, mouse, 1:1000, Sigma; alternatively, clone YL1/2, rat, 1:500, Millipore Bioscience Research Reagents); anti-DmEb1 (gift from H. Ohkura; rabbit, 1:2000; [28 (link)]); anti-GTP-tubulin (hMB11; human, 1:200; AdipoGen; [50 (link)]); anti-Shot (1:200, guinea pig; [51 (link)]); anti-Elav (Elav-7E8A10; rat, 1:1000; Developmental Studies Hybridoma Bank, The University of Iowa, IA, USA; [52 (link)]); anti-GFP (ab290, Abcam, 1:500); Cy3-conjugated anti-HRP (goat, 1:100, Jackson ImmunoResearch); F-actin was stained using phalloidin conjugated with TRITC/Alexa647, FITC or Atto647N (1:100 or 1:500; Invitrogen and Sigma). Specimens were embedded in ProLong Gold Antifade Mountant (ThermoFisher Scientific).