Imaging Drosophila Larval Muscle Development

The following Drosophila stocks were maintained at standard conditions on cornmeal medium: w¹¹¹⁸ (Bloomington 3605), Dmef2-GAL4 [48 (link)], UAS-2xEGFP (Blomington 6874), UAS-GFP RNAi (J. Zallen, SKI). Crosses (UASxGAL4) were performed at 25°C; embryos hatched within a 2h period were selected and raised to third instar larval stage. Wandering third instar larvae were dissected and fixed in 10% formalin and labeled as previously described [16 (link)]. Muscle cells were labeled using Alexa Flour-conjugated phalloidin (Life Technologies); nuclear DNA was visualized with Hoechst 33342 (Invitogen). Anti-Lamin (DHSB, ADL 67.10) and anti-α-Tubulin (Sigma, T9026) primary antibodies, and Alexa Flour-conjugated secondary antibodies (Life Technologies) were used to label the nuclei and microtubules, respectively. Whole larvae were mounted in ProLong Gold antifade reagent (Invitrogen). VL3 and VL4 muscles in abdominal hemisegments 2-6 were imaged on a LSM 700 confocal microscope (Zeiss).