For analysis of fixed samples mounted in PPDM (90% glycerol, 0.5% p-phenylenediamine, 20 mM Tris-HCl pH 8.8), images were acquired on a DeltaVision deconvolution Olympus IX70 microscope (Applied Precision) equipped with a CoolSnap CCD camera (Roper Scientific) at 20°C using a 100×, 1.35 NA Olympus U-Planapo oil objective lens. Immunofluorescence of fixed embryos was performed as described (Desai et al., 2003 (link)), using the following rabbit antibodies at a concentration of 1 μg/ml: α–CAR-1 (Cy3-labeled; described above); α–AIR-2 (Cy-5 labeled; generated against a GST fusion to the full-length protein); α–ZEN-4 (Cy-5 labeled; generated against a GST fusion to the COOH-terminal 108 aa); the mouse monoclonal antibody DM1α (Oregon green 488–labeled; Sigma-Aldrich); the goat polyclonal GFP antibody (Oregon green 488–labeled; generated against a 6x-histidine fusion to the full- length protein); and the unlabeled rat CGH-1 antibody (JDCR5; a gift of K. Blackwell, Joslin Diabetes Center, Boston, MA). For analysis of gonads, the tails of adult hermaphrodites were amputated in 5% sucrose and 100 mM NaCl to extrude the gonads. Fixation and immunofluorescence on gonads was performed as described for embryos. For live analysis, embryos were mounted on agarose pads as described previously (Oegema et al., 2001 (link)), and imaged on a spinning disc confocal microscope (Nikon Eclipse TE2000-E) equipped with a Hamamatsu Orca-ER CCD camera at 20°C using a Nikon 60×, 1.4 NA Planapo oil objective lens. For osmotically sensitive embryos and embryos imaged in the absence of compression, filming was performed in a depression slide containing meiosis media (25 mM Hepes at pH 7.4, 60% Leibowitz L-15 Media, 20% FBS, 500 μg/ml inulin) and sealed with petroleum jelly. Analysis of spindle pole separation, spindle microtubule density, and furrow movement was performed using Metamorph software.
Kymographs were constructed by compressing the image of the furrow region from each time point (same region as in Fig. 5 B) to a single vertical line, in which the maximum intensity along the x-axis of each original image is displayed for each point along the y-axis. The vertical strips for sequential time points are laid adjacent to each other so that time increases from left to right along the x-axis.