Embryonic Development Staging and In Situ Hybridization

Embryos were collected 0 to 3hr 30 min after the egg laying. Fixation of embryos and in situ hybridization with whole mount embryos was as described (Lu et al. 2008 (link)). Embryos are mounted in 70% glycerol in PBS for imaging. Stages of embryo were detected based on number of nuclei, shape of the nuclei, and cellular furrows as outlined (Lu et al. 2008 (link)). Templates for in vitro RNA transcription was made by PCR amplification with a forward primer and a reverse primer along with T3 promoter using genomic DNA from w¹¹¹⁸ flies. A Digoxygenin labeled antisense RNA probe was synthesized using in vitro transcription kit (MAXISCRIPT T3 kit, Ambion). Probe was detected using anti-Digoxygenin antibody (Roche) that cross react with NBT-BCIP solution staining the embryos. Primers used to in vitro templates were: Sxl forward 5′-CCCTACGTCGACGGCATTGCAGC-3′, Sxl reverse 5′-TAATACGACTCACTATAGG-GAATGACCCAATGGAATCG-3′ and runt forward 5′-AACGACGAAAACTACTGCGGCG-3′, runt reverse 5′-AATTAACCCTCACTAAAACGGTCACCTTGATGGCTTTGC-3′.