Immunostaining of Drosophila Testes and Embryos

Brains were dissected, squashed and stained as previously described (Stevens et al., 2009 (link)). Adult testes were dissected and fixed as described (Dix and Raff, 2007 (link)). Testes were then incubated with primary antibodies overnight at 4°C followed by washes with PBT and secondary antibody incubation for 4 hr at RT. Slides were washed in PBT and mounted for analysis. Embryos from 0–2 hr egg collections were aged for 1 hr at 25°C and were fixed and stained as previously described (Stevens et al., 2009 (link)). To preserve the GFP signal in embryos expressing either WT GFP-Sas-6 or GFP-Sas-6-F143D, embryos were fixed in 14.4% microfiltered FA solution containing 100 mM PIPES (pH 7.0), 2 mM EGTA and 1 mM MgSO₄ for 5 min. The following antibodies were used: sheep anti-Cnn (1:1000) (Cottee et al., 2013 (link)), guinea pig anti-Asl (1:500) (Cottee et al., 2013 (link)); GFP-Booster (ChromoTek, Germany) was used at 1/500 to enhance the GFP signal. Secondary antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 568 (Life Technologies) were used 1:1000. Hoechst33258 (Life Technologies) was used to visualise DNA.