Atto647n

Primary fly neurons and fibroblasts were fixed in 4% paraformaldehyde (PFA) in 0.05 M phosphate buffer (PB; pH 7–7.2) for 30 min at room temperature (RT); for anti-Eb1 staining, ice-cold +TIP fix (90% methanol, 3% formaldehyde, 5 mM sodium carbonate, pH 9; stored at −80°C and added to the cells; Rogers et al., 2002 (link)) was added for 10 mins. Adult brains were dissected out of their head cases in PBS and fixed with 4% PFA in PBS for 1 hr, followed by a 1 hr wash in PBT.
Antibody staining and washes were performed with PBT. Staining reagents: anti-tubulin (RRID:AB_477593, clone DM1A, mouse, 1:1000, Sigma; alternatively, RRID:AB_2210391, clone YL1/2, rat, 1:500, Millipore Bioscience Research Reagents); anti-DmEb1 (gift from H. Ohkura; rabbit, 1:2000; Elliott et al., 2005 (link)); anti-Elav (mouse, 1:1000, DSHB, RRID:AB_528218); anti-GFP (ab6673, goat, 1:500, Abcam, RRID:AB_305643); Cy3-conjugated anti-HRP (goat, 1:100, Jackson ImmunoResearch); F-actin was stained with Phalloidin conjugated with TRITC/Alexa647, FITC or Atto647N (1:100 or 1:500; Invitrogen and Sigma). Specimens were embedded in ProLong Gold Antifade Mountant.

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).

In order to optimize a sample preparation protocol for vimentin intermediate filaments in BHK21 cells, samples were prepared with a combination of different fixation, permeabilization and blocking methods. Two different primary antibodies, V9 (Sigma) and D21H3 (Cell Signaling Technologies) were applied to each sample preparation method. Two different secondary antibodies, Atto647N (Invitrogen) and Abberior Star635P (Abberior), were used as well (Supplementary Protocol 1). The samples were imaged with a Leica TCS STED (Leica Microsystems) super-resolution microscope. The STED microscopy image dataset contains a mixture of STED and confocal images, at various zoom levels; the STED images can be separated from the confocal images using the file filtering functionality in our PyImageQualityRanking software (see Image Quality Ranking Software).