Anti rabbit dylight 488

To acquire pupae of a defined age, white prepupae were collected at 0 h APF and aged on a moistened filter. Pupae or adult flies were dissected in a drop of phosphate-buffered saline (PBS; 0.13 mM NaCl, 7 mM Na₂HPO₄, 3 mM NaH₂PO₄) under a stereomicroscope. For antibody staining, tissues were fixed in 3.7% formaldehyde in PBS for 20 min, primary antibody was incubated overnight at 4°C and secondary antibody was added to the specimens at room temperature for 1 h. Hoechst 33258 (3 µg/ml; Sigma-Aldrich, 94403) was used to label DNA and Atto565-phalloidin (4 nmol/l; Sigma-Aldrich, 94072) was used to stain F-actin.
The following antibodies were used: anti-GFP (1:2000; Abcam, ab6556), anti-tropomyosin (1:1000; Abcam, ab50567), anti-phosphorylated histone H3 (Ser10) mitosis marker (1:500; Millipore, 06-570), anti-Mef2 (1:500; kindly provided by Hanh T. Nguyen, University of Erlangen, Germany), anti-Duf and anti-Rst (1:500 and 1:50, respectively; both gifts from Karl-Friedrich Fischbach, University of Freiburg, Germany), anti-β-Gal (1:1000; Biotrend, RGA1-45A-Z) and anti-Sns (1:250; generated by Pineda Antibody Service, Berlin). For fluorescent immunohistochemistry, the following secondary antibodies were used: anti-rat Alexa Fluor 488 (Jackson ImmunoResearch Laboratories), anti-rabbit DyLight488 (Vector Laboratories) and anti-guinea pig Cy2 (Jackson ImmunoResearch Laboratories).