Mouse anti galactosidase

Immunostaining of the wing discs was performed according to standard protocols. The following primary antibodies were used: rabbit anti-Phospho-histone 3 1:200 (Cell Signaling Technology, Danvers, Massachusetts, USA), rabbit anti-cleaved Dcp1 1:200 (Cell Signaling Technology, Danvers, Massachusetts, USA), mouse anti-Patched 1:500 (Gift from Isabel Guerrero), mouse anti-ß Galactosidase 1:200 (Promega, Madison, Wisconsin, USA), anti-ß Galactosidase 1:500 (Cappel, MP Biomedicals, Santa Ana, California, USA), rat anti-Phospho-Mad 1:100 (from Ginés Morata), and Rabbit Anti-Dpp (Gift from Matthew Gibson) [45 (link)]. Mouse anti-Repo and Rat anti-Elav 1:200 were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. Secondary antibodies (Molecular Probes, Oregon, USA) were used at dilutions of 1:200.
Imaginal discs were mounted in Vectashield mounting fluorescent medium (Vector Laboratories, Burlingame, California, USA).

Immunostaining was performed as described previously (Shlevkov and Morata, 2012 (link)). Images were captured with Leica TCS SPE and Zeiss LSM510 Vertical confocal microscopes. Images were processed with Fiji-ImageJ and Adobe Photoshop CS4 software using Student’s t for significance tests. Statistical analyses were performed with Microsoft Excel software. The following primary antibodies were used: rabbit anti-Caspase 3 (Roche, Basilea, Switzerland) 1:50; mouse anti-ß-Galactosidase (Promega, Madison, WI); rat anti-Cubitus interruptus (2A1; Hybridoma Bank, Iowa City, IA) 1:25; mouse anti-Engrailed (4D9; Hybridoma Bank) 1:50; mouse anti-Patched (Apa-1; Hybridoma Bank) 1:50; rabbit anti-H3K4me3 (07-473; Millipore, Billerica, MA) 1:50. Fluorescently labeled secondary antibodies (Molecular Probes, Eugene, OR) were used in a 1:200 dilution. TO-PRO3 (Invitrogen, Carlsbad, CA) was used in a 1:600 dilution to label nuclei; Phalloidin-Cy5 (65906; Sigma, St. Louis, MO) was used in a 1:200 dilution to label the F-actin network (thus the cell membranes).