Immunofluorescence Staining of Drosophila Ovaries

The following antibodies were used: mouse anti-Cut [Blochlinger et al., 1990 (link); Developmental Studies Hybridoma Bank (DSHB), 2B10], mouse anti-Hnt (Yip et al., 1997 (link); DSHB, 1G9), mouse anti-NICD (Fehon et al., 1990 (link); DSHB, C17.9C6), mouse anti-NECD (Diederich et al., 1994 (link); DSHB, C458.2H). All primary antibodies from DSHB were used at a dilution of 1:100. Anti-GM130 was purchased from Abcam (Sinka et al., 2008 (link); ab30637) and used at 1:500. Anti-Rab7 (rabbit) was kindly provided by the Nakamura lab and used at 1:1000 (Tanaka and Nakamura, 2008 (link)). AlexaFluor 488- and AlexaFluor 647-conjugated secondary antibodies were purchased from Jackson ImmunoResearch and used at a dilution of 1:1000 (115-545-003, 711-545-152, 115-607-020 and 111-605-003). F-actin was stained with Alexa Fluor 568 or Alexa Fluor 647 phalloidin (Invitrogen) at 1:1000. Ovaries were mounted in Vectashield with DAPI (Vector Laboratories). The cell membranes were labelled with CellMask Orange Plasma Membrane Stain or CellMask Deep Red Plasma Membrane Stain (Thermo Fisher Scientific).