α βgal

Dechorionated embryos were fixed in 4% formaldehyde, devitellinated with heptane/methanol, and antibody stained as described before (58 (link)). Antibodies used were α-Mef2 (1:1000, gift from R. Cripps, San Diego State University, San Diego, California, USA; generated as described in ref. 59 (link)), α–myosin heavy chain (1:600, Abcam, catalog MAC147), α-GFP (1:600, Torrey Pines Biolabs, catalog TP-401), and α-βgal (1:100, Promega, catalog Z3781). HRP-conjugated secondary antibodies (goat anti-mouse Fluor 488 catalog 115-545-166, goat anti-rabbit Fluor 488 catalog 111-545-003, and goat anti-rat Fluor 594 catalog 112-585-167, all Jackson ImmunoResearch) in conjunction with the TSA system (Molecular Probes) were used to detect primary antibodies.

For antibody staining, flies were dissected in Grace’s medium and ovaries were fixed and stained as described previously [44 (link)]. For LysoTracker staining, flies were dissected in PBS, incubated in 1:600 LysoTracker for 5 minutes, rinsed and washed in PBS, and finally fixed in PBS, heptane, and paraformaldehyde for 20 minutes. At this point, samples were treated as with standard antibody staining. All samples were mounted in VectaShield with DAPI (Vector Labs). Primary antibodies used were: cleaved α-Dcp-1 (1:100, Cell Signaling), α-Dlg (1:100, Developmental Studies Hybridoma Bank (DSHB)), α-Draper (1:50, DSHB), α-αPS3 (1:1000, [14 ]), and α-β-Gal (1:400, Promega). Secondary antibodies used were goat-α-rabbit Cy3 and goat-α-mouse Alexa Fluor 647 (Jackson ImmunoResearch), each at 1:100. Egg chambers were imaged on an Olympus FV10i confocal microscope, images were processed using ImageJ and Adobe Photoshop, and figures were made using Adobe Illustrator and Graphpad Prism.