For DAPI staining, ovaries were dissected in PBS and fixed in 4% electron microscopy (EM)-grade para-formaldehyde (Electron Microscopy Sciences) diluted in PBS for 30 min. Ovaries were counterstained with DAPI (Invitrogen) for 10 min. Embryonic cuticles and wings were prepared as described previously17 (link),18 (link). For immunofluorescence, ovaries were dissected from 3–5-day-old flies into ice-cold PBS and subsequently fixed in 4% formaldehyde (Thermo Scientific) containing 0.15% Triton X 100 (Sigma-Aldrich), diluted in PBS, for 25 min. After three rinses with PBT (PBS with 0.3% Triton X 100) ovaries were blocked in BBX (PBS containing 0.3% Triton X 100 and 0.1% BSA) for 30 min at room temperature (20–22 °C). Ovaries were incubated with primary antibodies over night at 4 °C diluted in BBX (antibodies to Piwi, Aub and Ago3, 1:500; antibodies to Armi and I element, 1:1,000; antibodies to Tudor, 1:10; antibodies to Spn-E, 1:50). After four PBT washes secondary antibodies were incubated 5 h at room temperature diluted in BBX (1:500; Molecular Probes). Ovaries were stained with DAPI for 10 min in the second of four PBT washes. Antibodies used were: antibody to Piwi, antibody to Aub and antibody to AGO3 (ref. 19 (link)); antibody to Tudor, antibody to Spn-E20 (link); antibody to Armi21 (link) and antibody to I element (gift from D. Finnegan; University of Edinburgh). For the sterility test, ten 3–5-day-old female flies were pre-mated with wild-type males overnight in small cages on apple juice plates with yeast paste. Apple juice plate was changed without anesthetizing flies. After 18 h at 25 °C, the flies were removed and the number of laid eggs was counted (typically ~200 eggs). Forty-eight hours later hatched and non-hatched eggs were determined.
Additional information on the phenotypic analyses of RNAi reagents is available inSupplementary Figures 7 and 8 as well as in Supplementary Table 3 .
Additional information on the phenotypic analyses of RNAi reagents is available in
