Anti β galactosidase

Protocol was described previously (Harmansa et al., 2015 (link)). Each fly cross was set together with control and >10 wing imaginal discs from each genotype were processed in parallel. If the genotype could be distinguished, experimental and control samples were processed in the same tube. A representative wing disc was shown for all the experiments. Following primary antibodies were used; anti-Dpp (1:100; Matthew Gibson), anti-phospho-Smad1/5 (1:200; Cell Signaling, 9516S), anti-Brk (1:1000; Gines Morata), anti-Wg (1:120; DSHB, University of Iowa), anti-Ptc (1:40; DSHB, University of Iowa), anti-β-Galactosidase (1:1000; Promega Z378A), anti-mCherry (1:5000; Nigg lab, University of Basel). All the primary antibodies except anti-Dpp antibody were diluted in 5% normal goat serum (NGS) (Sigma) in PBT (0.03% Triton X-100/PBS). Anti-Dpp antibody was diluted in 5% NGS in Can Get Signal Immunostain Solution B (TOYOBO). All secondary antibodies from the AlexaFluor series were used at 1:500 dilutions. Wing discs were mounted in Vectashield (H-1000, Vector Laboratories). Images of wing discs were obtained using a Leica TCS SP5 confocal microscope (section thickness 1 μm).

Fixation and staining of Drosophila imaginal discs was performed in ‘watch-glass’ containers. Wing discs were fixed in 4% paraformaldehyde (PFA) for 20 min, then washed twice in PBS for 20 min. A blocking step was carried out for 2 h at 25°C using 5% fetal calf serum in PBST (PBS+0.1% Triton X-100). Discs were incubated with Anti-β-Galactosidase (1:1000, Promega, Madison, WI, USA) and Anti-phospho-Histone H3 Ser10 (1:500, Cell Signaling Technologies, Danvers, MA, USA) primary antibodies overnight at 4°C (Hendzel et al., 1997 (link); Wang et al., 2010 (link)). Four 20-min washes were performed with PBST before incubation with secondary antibody. Alexa Fluor® (1:500, Invitrogen) secondary antibody was incubated for 2 h at 25°C. We washed four times 20 min washes in PBST and then a final wash of 20 min in PBS to remove detergent. Wing discs were mounted on microscopy slides with VECTASHIELD® Mounting Medium (Vector Laboratories). Slides were kept dark at 4°C to reduce fluorophore fading.

Lymph gland immunostaining was performed as previously described (Tokusumi et al. 2011 (link)). The following primary antibodies were used: mouse anti-Antp (1:100; 4C3, Developmental Studies Hybridoma Bank), anti-β-Galactosidase (1:100; Promega), and mouse anti-NimC1 antibody (Vilmos et al. 2004 (link)) (1:100; I. Ando). As secondary antibodies, we used the Alexa 555-conjugated anti-mouse IgG antibody (Invitrogen). We analyzed stained samples with a Zeiss Axioplan Fluorescence microscope or a Nikon AR-1 laser-scanning confocal microscope. Data were collected from at least 10 third instar larvae in all enhancer expression or gene phenotype analyses experiments.