Anti ct 2b10

The embryos were grown at 25°C except the 69B-Gal4 UAS overexpression experiments that were grown at 29°C to increase Gal4 activity. The following primary antibodies were used: mouse anti-AbdB 1A2E, anti-Crb and anti-Ct 2B10 (Developmental Studies Hybridoma Bank); rabbit anti-GFP (Invitrogen) and mouse anti-βGal (Promega). Secondary antibodies were conjugated to Alexa Fluor 488, 555 (Invitrogen). Confocal images were obtained with a Leica SP2-AOBS microscope and processed using ImageJ and Adobe Photoshop CS5. We used antisense RNA probes for upd, Gal4 and lacZ in situ.

Larval trachea was dissected at third larval instar and fixed in 4% formaldehyde for 20 min. Samples were incubated overnight at 4°C with primary antibodies and for 2 h at room temperature with secondary antibodies and then were mounted in Vectashield with DAPI (Vector Laboratories). The following primary antibodies were used: anti-Ct (2B10, 1:100) and anti-β-galactosidase (401.a, 1:200) from Developmental Studies Hybridoma Bank and anti-PH3 (1:500) from Cell Signaling Technology. Fluorescent-conjugated secondary antibodies were obtained from Molecular Probes. Images were obtained with SP5 confocal microscope and processed with either Fiji or Photoshop CS4 (Adobe).