SUPPLEMENTAL EXPERIMENTAL PRODECURES Antibodies

Rabbit polyclonal antisera directed against the N-terminal 14-16 AA of Piwi, Aub, and AGO3 were previously described (Brennecke et al., 2007). Primary antibody dilutions were 1:500 for immunohistochemistry. Anti-Vig antibodies (Caudy et al., 2002) were used at 1:2000 for western blots and anti-tubulin antibodies (Sigma B-5-1-2) at 1:5000. Secondary antibodies were Alexa Fluor ® (488, 568)-conjugated goat anti-rabbit and anti-mouse IgG (Molecular Probes, Invitrogen) for immunocytochemistry and anti-rabbit and anti-mouse HRP-conjugated IgG (Amersham Biosciences) for western blots. Immunocytochemistry Immunocytochemistry was done as previously described (Findley et al., 2003). Briefly, ovaries were fixed in 6.2 % formaldehyde for 5 minutes, rinsed three times in PBS, permeabilized for 10 minutes in PBS with 0.1 % Triton and washed three times in PBT (PBS plus 0.1 % Tween). Ovaries were then separated into ovarioles, blocked in 2 % BSA and incubated overnight at 4°C with primary antibodies. Ovarioles were then washed in PBT three times for 10 minutes