The Hedgehog (Hh) pathway is activated in follicle stem cells of animals lacking boi (boi[e]; this image), relative to wild-type Drosophila (CIL# 13756), based on activated cubitus interruptus (ci) staining (green). Follicle cells are highlighted with anti-Fas-3 (red) and germ cells are highlighted with anti-vasa (blue). Image is Supplemental Figure S3D 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 1:2,000 rabbit anti-Vasa (Hay et al., 1990); 1:25 mouse anti-Fas3 (Developmental Studies Hybridoma Bank [DSHB] and anti-activated ci. Secondary antibodies used were FITC, Cy3, and 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).
|Spatial Axis||Image Size||Pixel Size|