Immunostaining was performed and examined as previously described for S2 cells (Dzhindzhev et al., 2005 (link); Hughes et al., 2008 (link)), larval CNSs (Cullen et al., 1999 (link)), nonactivated oocytes (Cullen and Ohkura, 2001 (link)), activated oocytes (Cullen et al., 2005 (link)), and embryos (Cullen et al., 1999 (link)) except that an Axio Imager attached to an Exciter (LSM5; Carl Zeiss, Inc.) was used for confocal analysis. Orcein staining of larval CNS and phase-contrast microscopy of spermatids were described previously (Cullen et al., 1999 (link)). The frequency of aneuploidy in larval CNSs was estimated by orcein staining of squashed CNSs after incubation with colchicine (3 µg/ml in 0.7% NaCl) and hypotonic shock (in 0.5% sodium citrate). Squashed cells that clearly had six large chromosomes were counted as diploids, whereas cells with greater or less than six were counted as hyperploids or hypoploids, respectively. wacΔ and wild type showed 0.7 and <0.4% of hyperploids and 8.2 and 7.7% of hypoploids (n > 250), although most of the apparent hypoploids were likely to be caused by the squashing of brains (i.e., artifacts). Live S2 cells in the culture media on a concanavalin A–coated coverslip were examined at room temperature by a microscope (Axiovert; Carl Zeiss, Inc.) attached to a spinning-disc confocal head (Yokogawa) using Volocity (PerkinElmer). Immunostaining of centromere identifier showed equal segregation of centromeres in most spindles with telophase appearance in Wac-depleted S2 cells. We also see both genuine anaphase and pseudoanaphase cells with chromosomes scattered within a spindle. Intensity of GFP-tubulin signals was estimated by averaging the signal intensity within three equal-sized squares of 0.4 µm2 and subtracting the background signal. The intensities were normalized using the signal intensity at the poles in prophase. Signals of γ- and α-tubulin were measured in cells immunostained with GTU-88 and YOL1/34 (Sigma-Aldrich). For both signals, the mean intensity in the spindle (mean of three squares of 0.16 µm2) was normalized using the mean intensity at the pole after being background corrected. Significance was analyzed using the Wilcoxon test. For FISH in oocytes, stage 14 oocytes prepared as described previously (Cullen and Ohkura, 2001 (link)) were postfixed with 4% formaldehyde, and hybridization was performed as described in Dernburg et al. (1996) (link) using two Alexa Fluor 488–conjugated 40-mer oligonucleotides corresponding to the 359-bp repeats found at the X chromosome centromere as probes. Digital images were imported to Photoshop (Adobe) and contrast/brightness was changed uniformly across the field.