4 6 diamino 2 phenylindole dihydrochloride dapi

Prior to transfection, cells were seeded overnight onto 18-mm circular coverslips to achieve ~60% confluency. The cells were then transfected with egfp fusion plasmids using FuGENE® HD (Roche Applied Sciences) according to the manufacturer's protocol. After 18 h, the cells were fixed in 0.5 M PIPES buffer, pH 6.8 containing formaldehyde (3.7%) followed by staining with 1 unit of rhodamine-phalloidin (Life Technologies) and 0.35 µM 4,6-diamino-2-phenylindole (DAPI) dihydrochloride (Life Technologies). EGFP-positive cells were imaged using the Zeiss LSM510 META-UV confocal microscope and images were acquired and overlaid using Zeiss LSM Image Browser V4.2 software. For quantification, at least 50 visually rounded cells were counted per coverslip from at least 3 independent transfection experiments.
For transfection of FLAG-mcf-HA plasmids, HEK293T cells were seeded overnight in 6-well dishes followed by transfection with plasmids as described above.

The cells were fixed using ice-cold methanol for 6 mins or 4 % formaldehyde for 20 mins as indicated in the figures or figure legends. For Cdt1 spindle microtubule staining, cells were fixed in ice-cold methanol only for 3 mins. Following fixation, the cells were immune-stained with combinations of different primary antibodies, as indicated in the figures or figure legends. The primary antibodies used in the study include Hec1 monoclonal (1:400, ab3613, clone 9G3, Abcam), Zwint1 polyclonal (1:400, A300-781A, Bethyl), Tubulin monoclonal (1:500, T9026, clone DM1A, Sigma), anti-CREST antiserum (1:500, HCT0100, Immunovision, Inc.) and Cdt1 polyclonal (1:50, H300, Santacruz Biotechnologies). The Rabbit polyclonal antibody against Ska3 used at 1:200 dilution was a kind gift from Dr. Gary Gorbsky (Oklahoma Medical Research Foundation, University of Oklahoma). 4,6-diamino-2-phenylindole (DAPI) dihydrochloride (1:10,000, Life Technologies) was used to counterstain the nucleus/chromosomes. Alexa Fluor 488-, Rhodamine Red-X-, or Cy5-labeled donkey secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. and used at a dilution of 1:200.

The fixed brains were cryopreserved in 30% sucrose (wt/vol) in PBS and embedded in optimal cutting temperature compound. The olfactory tissues were cut on a cryostat into 20μm slices and stored at −80°C until use. The slices were pretreated for 30min in 0.025 M HCl at 65°C and rinsed with 0. 1M borate buffer (pH 8.5), PBS and TBS-T (10mM Tris-HCl (pH 7.4), 100 mM NaCl with 0.3% Triton X-100 (vol/vol)). The slices were then blocked with blocking buffer (5% normal donkey serum (vol/vol) in TBS-T) at 20 – 25°C for 1 hour and incubated with primary antibodies diluted in blocking buffer overnight at 4°C. Sections were washed with TBS-T and then incubated with secondary antibodies with 4’6-diamino-2-phenylindole dihydrochloride (DAPI; Thermo Fisher Scientific; Waltham, MA; RRID:AB_2629482) for nucleus staining for 1 hour. The immunoreacted sections were washed and coverslipped with Fluoro-Gel mounting medium (Electron Microscopy Science; Hatfield, PA).
Primary antibodies used in this study are summarized in Table 1. Goat or donkey anti-species IgG conjugated with Alexa 488 or Alexa 555 (Thermo Fisher Scientific; 1:300; RRID:AB_141708, RRID:AB_162543, RRID:AB_141514, RRID:AB_141596, RRID:AB_141709, RRID:AB_142672, RRID:AB_141788) were used as secondary antibodies.