Cy3 conjugated monoclonal donkey anti mouse igg

BrdU incorporation and labelling was performed essentially as previously described (http://theduroniolab.web.unc.edu/files/2013/10/Eye-disc-BrdU.pdf). 5′ bromodeoxyuridine was fed to 120-hour-old larvae at a concentration of 0.1 mg/ml. Wing discs were dissected, fixed in 4% formaldehyde and permeabilised for 45 min in PBS + 0.6% Triton X-100. Discs were incubated in 2N HCl for 30 min at room temperature and then neutralised in sodium borate. Discs were washed 3 times in PBS + 0.3% Triton X-100 before incubation in anti-BrdU overnight at 4°C (Developmental hybridoma bank (G3G4), 1:20). The secondary antibody was Cy3-conjugated monoclonal Donkey anti-mouse IgG (Jackson ImmunoResearch, cat. no.715-165-151; 1:350).

Immunocytochemistry was performed essentially as described in (Sullivan et al., 2000 ). Images were taken with a Zeiss Axiovert confocal microscope equipped with a LSM520 Meta. Primary antibodies used were anti-Pacman (Grima et al., 2008 (link)) (1:500), anti-Cleaved Caspase-3 (Asp175) (Cell Signaling, cat no. 9661;1:400), anti-phosphohistone H3 (Ser10) (Cell Signaling, cat. no. 9701; 1:400) and anti-Wingless (4D4) (Developmental Studies Hybridoma Bank; 1:400). Secondary antibodies used were Cy3-conjugated monoclonal goat anti-rabbit IgG (Jackson ImmunoResearch, cat. no. 711-165-152; 1:400), Cy3-conjugated monoclonal Donkey anti-mouse IgG (Jackson ImmunoResearch, cat. no.715-165-151; 1:400) and FITC-conjugated polyclonal goat anti-rabbit (Sigma, cat. no. F9887; 1:200).