Embryos from the appropriate crosses were collected on grape plates for four hours and after hatching, larvae were raised on mounds of yeast paste on grape plates at 22oC. For crosses involving btl-Gal4, UAS-Actin-GFP/CyO, GFP larvae were selected using a Leica MX FluoIII fluorescence microscope. For imaging, larvae were immobilized either with ether vapor before mounting in 70% glycerol or by heating for a few seconds on a 70oC hot plate [26 (link)] after mounting. Microscopes used for imaging were as follows; bright field and fluorescence images in Fig 1 (A-D, F, G) and Fig 5 (G, H)—Zeiss Axioplan2; bright field images in Fig 5 (A-D)—Zeiss Axioimager 2; bright field images in Fig 1(E) and Fig 5 (E, F)—Zeiss Axioskop. For quantitation of tracheal defects (Fig 5 ), larvae were imaged from Day1 after hatching until the day before pupation or death, as determined form the studies in Figs 3 and 4 . At least 10 larvae were imaged for each genotype and fluid-filling of the tracheae was quantitated from stored image sets.
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