Larvae are mounted lateral side down in 1% low gelling temperature agarose dissolved in PBS, over glass bottom culture dishes and overlaid with PBS for imaging. Use a flexible Eppendorf Microloader Pipette Tip to orient larvae in a lateral position for better visualization of fluorescent reporter expression. Z-stack acquisition is performed with Zeiss Zen 3.0 (blue edition) software for multipositions in an automated and motorized inverted microscope (Zeiss Axio Observer) using a 10× objective (NA 0.3) and a mercury lamp. Position the larvae to have the more distal part of the intestine centered anterior posteriorly and dorsal ventrally in the image. Set the lumen of the gut as the z-center level. Image each larva in the channels brightfield and mCherry (ex: 585 nm, em: 610 nm band pass filters) in 49 focal planes (149 μm range; 3 μm z step) so that all the gut depth is visible.

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