Deltavision image restoration microscope

Embryos at late ball stage were mounted on a 5% agar pad without azide and were washed repeatedly to remove bacteria before mounting. Images were collected using a Weatherstation environmental chamber set at 20°C on a DeltaVision Image Restoration Microscope (Applied Precision) with an Inverted Olympus IX-70 microscope, and using a 63x silicone oil-immersion objective and a Photometrics CoolSnap HQ camera (Roper Scientific). Time-lapse images were acquired until the embryo began to twitch at early 1.5-fold stage. A stack of optical sections at 0.5 um spacing was acquired at 12 min intervals, using conditions for non-saturated signal, 10-32% power, and exposure times of 0.2-0.5 sec, depending on the labeling reporter used. Image acquisition did not result in appreciable developmental arrest. Between time points the focal midpoint of the stack was adjusted to compensate for rotation of the embryo and movement of the cell of interest. Deconvolution of DeltaVision Images was performed with Softworx (Applied Precision).

All experiments were performed on a Zeiss Axioplan2 microscope (Carl Zeiss MicroImaging), with a 40x objective (Plan Neo, NA 0.75) and DeltaVision Image Restoration Microscope (Applied Precision). HCT116 cells were plated on glass coverslips (Fisher Scientific) in 6-well dishes 24 hours before fixation. Cells were exposed to DMSO only (vehicle control), ispinesib (50 and 100 nM) for 4 hours, or 50 ng/mL (166 nM) nocodazole for 14 hours at 37 °C in complete media. Cells were fixed for 10 min at 37°C in fix solution (4% formaldehyde, 0.2% Triton X-100, 10 mM EGTA, 1 mM MgCl2, 100 mM PIPES pH 6.8). Coverslips were washed 3 times with TBS-tx (TBS + 0.1% Triton X-100), blocked with AbDil (2% BSA in TBS-tx buffer) and incubated for 1 hr at room temperature with FITC-conjugated mouse anti-tubulin monoclonal antibody (Sigma # F2168; 1:2000 dilution in AbDil). Coverslips were washed three times in TBS-tx, and DNA was stained with Hoechst 33342 (Sigma; 1:10,000). Coverslips were mounted in 0.5% p-phenylenediamine (Sigma) in 20 mM Tris, at pH 8.8, with 90% glycerol and sealed with nail polish.