Midgut Dissection and Immunostaining

Intact female midguts were dissected using standard protocols^{59 (link)}. Primary antibodies were: anti Dronc^{60 (link)} (1:200; a kind gift of Pascal Meier); PH3 (1:2,000; Millipore), Prospero (Pros, 1:20; DSHB; Prospero (MR1A) was deposited to the DSHB by C.Q. Doe); Pdm-1^{61 (link)} (1:1,000; a kind gift of Yu Cai). DAPI was used to counterstain nuclei. Phalloidin labeling was used to assess the physical properties of the guts. Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch. If not noted otherwise, region R4ab^{35 (link),36 (link)} in the posterior midgut was imaged and analysed. Images were obtained with a Zeiss LSM 700 confocal microscope, analysed with Zen 2012 imaging software (Carl Zeiss) and processed with Adobe Photoshop CS6.