Adult lenses were visualized by SEM using anesthetized flies mounted on carbon tabs and directly analyzing them with a Hitachi S-3400N. For thin sections, eyes were fixed for 15 minutes in 4% paraformaldehyde/phosphate-buffered saline, washed twice with 0.1% Triton X/phosphate-buffered saline (PBT), serially dehydrated in ethanol, incubated in 1:1 ethanol/LR-White resin (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hour and 100% resin for 1 hour, then polymerized with one drop per milliliter of accelerator. Sections (2 μm) were dried for 15 minutes on a slide warmer and stained with 1% toluidine blue for 10 minutes and mounted in Entellan (EMS), or rehydrated in PBT overnight followed by antibody staining and mounting as previously described [74 (link)]. Imaginal discs and retinas dissected from pupa 45 hours after puparium formation (45% pupation) were immunostained as previously described for whole mount adult retinas [74 (link)]. Antibodies were from the Developmental Studies Hybridoma Bank unless indicated otherwise, and diluted as follows: Cut (mouse, 1:100), Pros (rabbit [50 (link)], 1:1,000), Pros (mouse, 1:10), Pros (guinea pig, this paper, 1:1,500), dPax2 (rabbit [28 (link)], 1:50; rabbit, this paper, 1:1500; guinea pig, this paper, 1;1,500), Elav (mouse or rat, 1:200), Eya (mouse, 1:50), BarH1 (rat, this paper, 1:200; rabbit [38 (link)], 1:50), Dlg (mouse, 1:50), E-cadherin (rat, 1:20; rabbit, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, 1:50), GFP (chicken, Abcam, Cambridge, MA, USA, 1:500), β-gal (chicken, Sigma-Aldrich, St. Louis, MO, USA, 1:1,000), Otd (guinea pig [74 (link)], 1:750), Rh3 (chicken [32 (link)], 1:40), Rh4 (rabbit, gift from C Zuker/N Colley, 1:150), Delta (mouse, 1:50), E(spl) (mouse [44 (link)], 1:1), pERK (rabbit, Sigma-Aldrich, 1:20), m(Espl) (1:2 [44 (link)]) and N-Cad (rat, 1:20). Secondary antibodies were conjugated to Alexa Fluor 488, 555, 647 and 750 nm (goat, Invitrogen) or Cy2, 3 or 5 (donkey, Jackson Immunoresearch, West Grove, PA, USA), and diluted 1:500. Polymerized actin was detected using AlexFluor 488-conjugated phalloidin (Invitrogen), which was added to the secondary antibody dilutions at 1:20 according to the manufacturer's suggestion. Samples were imaged with a Zeiss Apotome, deconvolved with Axiovision 4.6 or Zeiss LSM 700 confocal and processed in Adobe Photoshop 7.0.
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