Fly ovaries were dissected and fixed as described previously (O’Reilly et al., 2008). For anti-Boi immunostaining, ovaries were fixed in 2% formaldehyde on ice. Primary antibodies were 1:50 rat anti-Boi (Hartman et al., 2010); 1:100 goat anti-Hh (Santa Cruz Biotechnology, Inc.) or 1:100 rabbit anti-Hh (Ballet et al. 2003). Secondary antibodies used were FITC, Cy3, and/or Cy5 conjugated to species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). Samples were mounted in Vectashield mounting medium. Images were collected at room temperature (22C) using 40× (1.25 NA) or 63× (1.4 NA) oil immersion lenses (Leica) on an upright microscope (DM 5000; Leica) coupled to a confocal laser scanner (TCS SP5; Leica). LAS AF SP5 software (Leica) was used for data acquisition. Images representing individual channels of single confocal slices from the center of each germarium were exported as TIFF files, and images were converted to figures using Photoshop software (Adobe).