Quantitative assessment of Stat92E-GFP expression and anti–p-Stat staining during oogenesis

For anti-phospho-Stat antibody staining, female flies were dissected in dissection medium containing 1× phosphatase inhibitors (PhosSTOP, Sigma-Aldrich), and fixed (4% paraformaldehyde, 1X PBS and 1× phosphatase inhibitors) for 2 hours at 4℃. Finally, samples were washed once with 0.5% PBT then washed again with 0.3% PBT before primary antibody treatment. The anit-phospho-Stat antibodies (1:400) were prepared in 0.3% PBT and incubated with samples overnight at 4℃. The following secondary antibodies, conjugated with fluorescent dye were used at 1:400 dilutions: Alexa-488, Alex-568 or Alexa-647 (Invitrogen, Molecular Probes).