α dlg

Adult female flies were dissected in PBS. All the digestive tract was removed and fixed in PBS and 4% electron microscopy grade paraformaldehyde (Polysciences, USA) for 35 minutes. Samples were rinsed 3 times with PBS, 4% BSA, 0.1% Triton X-100 (PBT-BSA), incubated with the primary antibody overnight at 4°C and with the secondary antibody for 2 hours at room temperature. Finally, the samples were rinsed 3 times with PBT-BSA and mounted in DAPI-containing media (Vectashield, USA). All the steps were performed without mechanical agitation. Primary antibodies mouse α-Pros (1:100), α-Dl (1:10), α-Arm (1:10), α-Dlg (1:250) and α-Sn (1:50) were obtained from the Developmental Studies Hybridoma Bank (DSHB), rabbit α-PH 3 (1:100) from Cell Signalling (USA), rabbit α-Pdm1 (1:1000) was a gift of Dr.Yang Xiaohang (Institute of Molecular and Cell Biology, Singapore), rabbit α-laminin (1:1000) was from Sigma (USA) and goat α-GFP (1:500) was form Abcam (UK). Secondary antibodies were from Invitrogen (USA). TRICT-conjugated Phalloidin (Sigma, USA) was used at 5 µg/ml. Images were obtained on a Leica SPE or Leica SP5 confocal microscopy and processed in Photoshop CS5 (Adobe, USA).

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.