Rabbit anti β galactosidase

Dissected wing imaginal discs were fixed and stained with antibodies according to standard procedures. The following primary antibodies were used: rabbit anti-pMad, 1:200 (Cell Signalling); mouse anti-α-Tubulin, 1:2000 (Sigma); rabbit anti-β-galactosidase, 1:2000 (Promega); mouse anti-Ubx, 1:200 (DSHB); rabbit anti-Omb, 1:1000 and rabbit anti-Sal, 1:500 (a gift from Coralia Pérez Fernández); mouse anti-EGFR, 1:200 (abcam); and mouse anti-Wg 1:200 (DSHB). Secondary antibodies (diluted 1:200) included goat anti-mouse DyLight 488 and goat anti-mouse DyLight 549 (Agrisera) and goat anti-rabbit DyLight Cy5 (Jackson ImmunoResearch). Cell nuclei were stained with DAPI (1:500, Sigma). F-actin was visualized with Rhodamine-phalloidin, 1:2000 (Sigma). Images were collected using a Leica TCS SP2 AOBS confocal microscope. Cell height was calculated based on high resolution confocal images using the Image-J program.

The intestines from female adults were dissected in cold PBS and fixed immediately in PBS containing 4% (weight/vol) paraformaldehyde. Samples were then washed in PBS with 0.1% Triton X-100 (vol/vol) three times, blocked in PBTB [PBT containing 5% (vol/vol) normal goat serum], and incubated with primary antibodies overnight. The following primary antibodies were used: rabbit anti-Clbn (1:400), mouse anti-Prospero (1:200, Developmental Studies Hybridoma Bank), rabbit anti-Pdm1 (1:400), mouse anti-Armadillo (1:50, Developmental Studies Hybridoma Bank), rabbit anti-β-galactosidase (1:200, Promega), rabbit anti-phospho–histone-H3 (1:200, Millipore), rabbit anti-H2AvD pS137 (1:200, Rockland), rabbit anti-dpERK (1:200, Cell Signaling Technology), Alexa-Fluor-555-conjugated Phalloidin (1:500, Thermo Fisher Scientific). After three washes with PBT, secondary anti-rabbit or anti-mouse fluorescence antibodies, including Alexa 488 and 555 (1:400, Cell Signaling Technology), were used. Samples were mounted and analyzed on a Olympus FV1000 confocal laser-scanning microscope. The images were processed using Adobe Photoshop, Illustrator, and ImageJ.

Fixed wing imaginal discs were stained with antibodies according to standard procedures. The primary antibodies used were: rabbit anti-Caspase3, 1:200 (Cell Signaling Technology); mouse anti-β-galactosidase, 1:2000 (Promega); rabbit anti-β-galactosidase 1:2000 (Promega); rat anti-Ci, 1:200 (DSHB); mouse anti-En, 1:200 (DSHB); mouse anti-Smo, 1:200 (DSHB); mouse anti-Ptc, 1:200 (DSHB). Secondary antibodies used were goat anti-mouse DyLight 549, goat anti-rat DyLight 549, and goat anti-rabbit DyLight 488, 1:200 (Agrisera). Images were collected using a Leica TCS-SP2-AOBS confocal microscope.