Rat anti de cadherin

For immunolabeling pupal eyes, 40–42 h after puparium formation, were dissected in PBS and fixed in 4% formaldehyde. Fixed tissues were washed three times in PBS solution containing 0.1% Triton-X-100 and blocked in 5% normal goat serum for 1 h before incubation with primary antibodies. The primary antibodies used in this study were rabbit anti-Magi 1:200^{39 (link)}, rabbit anti-Baz 1:1000, rat anti-DE-cadherin 1:50 (Developmental Studies Hybridoma Bank). The appropriate secondary antibodies were conjugated Alexa488, Alexa594, and Alexa 647 (Invitrogen).

Wandering 3rd-instar larval wing discs were dissected in PBS, fixed in 4% paraformaldehyde diluted in PBS for 40 min, washed once for 5 min in PBX (PBS with 0.1% Triton X-100), twice for 20 min in PAXD (PBS with 1% bovine serum albumin, 0.3% Triton X-100, and 0.3% deoxycholate), and once in PAXDG (PAXD with 5% normal goat serum), all on ice. The tissues were incubated overnight in primary antibody diluted in PAXDG at 4°C and washed three times in PBX at room temperature. After >4 h of incubation in secondary antibody diluted in PAXDG at 4°C, they were washed twice in PBX and once in PBS, all at room temperature. The prepared tissues were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The antibodies used were rat anti-DE-cadherin (1:20; from the Developmental Studies Hybridoma Bank at the University of Iowa) and rabbit anti-β-galactosidase (1:2,000; ICN/Cappel); the secondary antibodies were conjugated to Alexa Fluor 568 (Invitrogen) and Cy5 (Jackson ImmunoResearch). Immunofluorescence was acquired on a Zeiss LSM 510 confocal microscope. Image J64 (National Institutes of Health, Bethesda, MD) was used to adjust the brightness and contrast of whole images.