Goat anti mouse igg alexa fluor 405

Cells synchronized with the Cdk1 inhibitor RO-3306 were released with a drug washout and monitored for mitotic entry. Cells were fixed with 4% paraformaldehyde (15 minutes) after ~ 1 hour after drug washout, where the maximal number of cells were observed (by visual inspection) to be in metaphase. Fixed cells were permeabilized in 0.1% Triton X-100 solution for 15 minutes and blocked with 5% goat serum (Invitrogen, Grand Island, NY) for 45 minutes. Primary antibodies were diluted in an antibody dilution buffer consisting of PBS with 1% Bovine Serum Albumin and Triton-X 100 and stored overnight at 4° C. Primary antibodies include Anti-beta tubulin (1:500, mouse monoclonal, 2 28 33, Invitrogen) and Anti-Hec1 (1:1000, human monoclonal) for labeling microtubules and kinetochores respectively. Secondary antibodies diluted in antibody dilution buffer were added along with the conjugated Phalloidin-TRITC (Santa Cruz Biotechnology) or Alexa Fluor 647 Phalloidin (1:40–1:80, Invitrogen) and stored in a dark place for 45 minutes. Secondary antibodies include donkey anti-human IgG Alexa Fluor 555 (1:600) and Goat anti-mouse IgG Alexa Fluor 405 (1:500, Invitrogen). Confocal microscopy was performed using a laser scanning confocal microscope (LSM 880, Carl Zeiss Inc.) with optimal imaging settings and z-slice thicknesses ranging from 0.36–0.5 μm.

Cells were fixed with 4% paraformaldehyde for 15 min. Following 2 times PBS wash, permeabilization was performed with a 0.1% Triton X-100 solution. Permeabilized cells were washed with PBS (2×) and blocked with 5% goat serum (Invitrogen, Grand Island, NY) for 45 min. Primary antibodies were diluted in an antibody dilution buffer consisting of PBS with 1% bovine serum albumin and Triton X-100 and stored overnight at 4 °C. Primary antibodies include anti-β-tubulin (1:500, mouse monoclonal, 2 28 33, Invitrogen), anti-Hec1 (1:1,000, human monoclonal), and anti-phospho-paxillin (1:100, rabbit polyclonal, pTyr31, Invitrogen). Secondary antibodies diluted in antibody dilution buffer were added along with the conjugated Phalloidin-TRITC (Santa Cruz Biotechnology) or Alexa Fluor 647 Phalloidin (1:40 to 1:80, Invitrogen) and stored in a dark place for 45 min. Secondary antibodies include donkey anti-human IgG Alexa Fluor 555 (1:600), goat anti-mouse IgG Alexa Fluor 405 (1:500, Invitrogen), and goat anti-mouse IgG Alexa Fluor 647 (1:500, Invitrogen). Confocal microscopy was performed using a laser scanning confocal microscope (LSM 880, Carl Zeiss Inc.) with optimal imaging settings and z-slice thicknesses ranging from 0.36 to 0.5 µm.

For cryosection staining of cytoplasmic dynein-1 components, eyes from light-adapted adult (2 mo) zebrafish were enucleated and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). For cryosection staining of phalloidin, whole 7 dpf embryos that were injected with TαCP:tdEOS-tubulin for mosaic expression were fixed in 4% PFA in PBS. Following fixation, samples were infiltrated with sucrose and embedded in OCT freezing medium before being cut into 12-μm sections and subsequently stained. For ex vivo CIS–COS staining, isolated CIS–COS from TαCP:tdEOS-tubulin fish were fixed in 4% PFA in PBS and subsequently stained. The Dync1H1 (20928), total Dynein IC (V3), and Dync1I2 (IC2; Vaughan and Vallee, 1995 (link); Vaughan et al., 2001 (link); Whyte et al., 2008 (link)) antibodies were gifts from Richard Vallee (Columbia University, New York, NY) and Kevin Vaughan (University of Notre Dame, Notre Dame, IN). Adherens junctions and the actin cytoskeleton were stained with Alexa Fluor 594 phalloidin (Invitrogen, Waltham, MA). Basal bodies were stained with GTU-88, a mouse monoclonal anti-γ-tubulin (Abcam), and rabbit polyclonal anti-centrin1 (C7736; Sigma) antibodies. Secondary antibodies included goat anti-rabbit immunoglobulin G (IgG) Alexa Fluor 594 and goat anti-mouse IgG Alexa Fluor 405 (Invitrogen).