Hedgehog (Hh, red) is transcribed in apical cells of the Drosophila wild-type germarium (CIL# 13755, 13744). In the absence of boi (boi[e] mutant), Hh protein is redistributed from apical cells to the extracellular space of the local follicle stem cell niche (CIL# 13745), but Hh transcription is still in apical cells (this image; boi[e]; Hh-lacZ). Hh transcription is demonstrated by the Hh-lacZ reporter and follicle cells are highlighted using anti-Fas-3 (green). Image is Supplemental Figure S3B in J Cell Biol. 2010. 191: 943-952.
Fly ovaries were dissected and fixed as described previously (O’Reilly et al., 2008). Primary antibodies were anti-betagalactosidase and 1:25 mouse anti-Fas3 (Developmental Studies Hybridoma Bank [DSHB]. Secondary antibodies used were either 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).
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