Fig. 2. Acridine Orange staining of third-instar intestines. (A) A segment of wild-type gut stained with AO. The CC apical invaginations (arrows) and gut lumen (L) are filled with a strongly red-shifted AO signal indicating robust acid production. The interstitial cells forming the pores also contain a ring of green fluorescence of unknown origin (arrowheads). (B-D) Wild-type third-instar CC; (B), brightfield view; (C), AO fluorescence. Parasagittal section showing red fluorescence in the apical invagination and pore, whereas the section intersects a green fluorescent ring in the interstitial cells. (D) Illustration of the efficacy of the AO probe by showing the relative red and green channel fluorescence along a transect in the cell in D (see inset). (E) Low-magnification views of representative wild-type guts stained with AO. All show a strongly red-shifted signal in the CC apical invaginations and gut lumen. The first panel is parasagittal, the rest are sagittal. L, lumen. (F) Low-magnification views of representative karst guts stained with AO. There is no AO signal from the CC apical invaginations, whereas the lumens are green to pale orange. Strong accumulation of red-shifted AO is seen in vesicular structures in mutant CCs. The first panel is parasagittal and the rest are sagittal. L, lumen. (G) Higher-magnification view of the segment of karst gut stained with AO in the rightmost panel in F, showing how the CC apical invaginations (arrows) are devoid of AO staining. The gut lumen is filled with mostly unshifted signal, indicating weak acidification. No green rings appear near the pores. Prominent red-shifted vesicles indicating low pH are visible in the CC cytoplasms (e.g. arrowheads). L, lumen. Bars, 20 µm.