A splice-blocking MO targeting the exon 2–intron 2 boundary of c9hxorf36 was designed and obtained from Gene Tools (5′-AAGCCACAAATTCAGACCTTCACCA-3′). The efficiency of the MO was confirmed by RT-PCR (reverse transcription–polymerase chain reaction). RNA was extracted from MO-injected embryos and controls at 3 dpf using TRIzol (Invitrogen). First-strand cDNA was synthesized using SuperScript III reverse transcriptase and random hexamers (Invitrogen), according to the manufacturer's instructions. We carried out PCR amplification using whole-embryo cDNA as template, and PCR products were subjected to direct DNA sequencing (Supplemental Fig. S2). One nanoliter of diluted MO (3, 6, and 9 ng) was injected into zebrafish embryos at the one- to four-cell stage. Injected embryos were fixed in 4% PFA (paraformaldehyde) overnight at 5.5 dpf (Alcian blue staining) and at 2 dpf for phospho-histone H3 staining. Alcian blue staining was performed to demarcate cartilage structures as described previously (Chassaing et al. 2012 (link)). Phospho-histone H3 immunostaining was performed as described previously (Golzio et al. 2012 (link)) using anti-histone H3 (ser10)-R, (sc-8656-R, Santa Cruz; 1:500 dilution). Embryos were imaged on a Nikon AZ100 microscope facilitated by NIS Elements software.