Phalloidin alexa fluor 488 647

Wing discs from third-instar larvae were dissected and fixed for 15 min at room temperature in 4% paraformaldehyde in PBS. Washing steps were performed in PBS containing 0.1% TritonX-100 (PBT). Discs were then incubated with primary antibodies in PBT, gently mixing overnight at 4°C. Tissues were counterstained with DAPI (0.25 ng/µl, Sigma, D9542), Phalloidin-Alexa Fluor 488/647 (1:100, Life Technologies) or Phalloidin-conjugated TRITC (1:400, Sigma) during incubation with cross-absorbed secondary antibodies coupled to Alexa Fluorophores (Invitrogen or Abcam) at room temperature for 2 hr. The gstD-GFP is a GFP reporter under the control of 2.7 kB upstream regulatory region of GstD as published by Dirk Bohmann’s lab (Sykiotis and Bohmann, 2008 (link)) and the Trbl GFP is a GFP-trap MIMIC line inserted in the intron of Trbl (Bloomington stock #61654). Tissues were mounted using SlowFade Gold Antifade (Invitrogen, S36936). Whenever possible, experimental and control discs were processed in the same vial and mounted on the same slides to ensure comparability in staining between different genotypes. Images were acquired using the Leica TCS SP8 Microscope, using the same confocal settings and processed using tools in Fiji. Per-channel views are shown in Figure 3—figure supplement 5.

Wing discs from third instar larvae were dissected and fixed for 15 min at room temperature in 4% paraformaldehyde in PBS. Washing steps were performed in PBS containing 0.1% TritonX-100 (PBT). Discs were then incubated with primary antibodies (described in S1 Table) in PBT, gently mixing overnight at 4°C. Tissues were counterstained with DAPI (0.25 ng/μl, Sigma, D9542), Phalloidin-Alexa Fluor 488/647 (1:100, Life Technologies), or Phalloidin-conjugated TRITC (1:400, Sigma) during incubation with cross-absorbed secondary antibodies coupled to Alexa Fluorophores (Invitrogen or Abcam) at room temperature for 2 h. Tissues were mounted using SlowFade Gold Antifade (Invitrogen, S36936). Whenever possible, experimental and control discs were processed in the same vial and mounted on the same slides to ensure comparability in staining between different genotypes. Images were acquired using the Leica TCS SP8 Microscope, using the same confocal settings and processed using tools in Fiji. Of note, all wing discs expressing the 10xStat92E-dGFP reporter were boosted with an anti-GFP antibody.