Dmra2 widefield epifluorescence microscope

For quantification of ploidy in Figure 3, wing imaginal discs from Tb+ 3^rd instar larvae were dissected and fixed as described (Schwed et al. 2002 (link)). Discs were labeled with rabbit anti-dsRed (Clontech, 632496) (1:400) and secondary anti-rabbit Alexa Fluor 568 (1:500) (Invitrogen), and stained with DAPI (0.5μg/ml). Discs were imaged on a Leica SP5 confocal and Leica DMRA2 widefield epifluorescence microscope. ImageJ was used to quantify nuclear area and total DAPI fluorescence. The nuclear area and DAPI intensity of cells within the RFP+ dpp expressing stripe were normalized to cells outside of the stripe in the same wing discs (RFP + cells / RFP- cells in Figure 3). The cells in the wing pouch area of each wing disc were scored, excluding the zone of non-proliferating cells, which are arrested in G1 and G2 phases of the cell cycle (Johnston and Edgar 1998 (link)).

BrdU, antibody and DAPI labeling for Drosophila ovaries were performed as previously described (Calvi and Lilly 2004 ). α-BrdU (mouse monoclonal, BDB347580; BD Biosciences, San Diego, CA) was used at 1:20. α-GFP (rabbit, Molecular Probes, Life Technologies), and secondary antibodies Alexa 488 anti-rabbit (Invitrogen) were used at 1:500. For EdU labeling, unfixed cells were incubated in 10 µM EdU followed by fixation with formaldehyde. EdU incorporation was detected with Alexa Fluor-488 Azide (Invitrogen) in a copper-catalyzed Click-iT reaction (Buck et al. 2008 (link); Salic and Mitchison 2008 (link)). Images were taken with a Leica DMRA2 widefield epifluorescence microscope (Figure S2, and Figure S5, C and D), and a Leica SP5 laser scanning confocal microscope (Figure 2, Figure 6, Figure S1, Figure S4, and Figure S5, A and B).