After freeze-cracking, embryos were fixed in −20°C methanol for 15 min and then incubated with 1:1,000 rabbit anti–KLP-7, a gift from K. Oegema at the University of California, San Diego, La Jolla, CA (Oegema et al., 2001 (link)), and 1:50,000 mouse anti–α-tubulin (DM1A; Sigma-Aldrich) antibodies at 4°C overnight. As previously described, the KLP-7 antibody was generated by amplifying a fragment of KLP-7 from cDNA using the primers 5′-CGCGCGAGATCTCAGAGAAAACGAGCCGAGAA-3′ and 5′-GCGCGCGAATTCTCAAGGAGCCATACGAACAGGAAC-3′. Fragments were digested with BglII-–EcoRI and cloned into pGEX6P-1 (GE Healthcare) to produce GST-KLP-7 (Oegema et al., 2001 (link)). After washing, the slides were incubated with 1:1,000 anti-rabbit–Alexa 488 and 1:1,000 anti-mouse–Alexa 555 (Life Technologies) for 2 h at room temperature. Slides were then incubated with 100 ng/ml DAPI (Roche) for 15 min before mounting. Images were collected with a confocal microscope (LSM 700; Carl Zeiss), and a z projection is shown in Fig. 1 D. A predicted but unverified in-frame splice product of klp-7(tm2143) would remove amino acids 97–364 (Fig. 1 and see the last paragraph of the preceding subsection); the peptide used as the antigen for generating the KLP-7 antisera included amino acids 14–190, and thus, the antisera might not recognize an in-frame deletion allele protein product (Oegema et al., 2001 (link)).