Mouse anti β galactosidase

For antibody staining, the lymph glands were dissected in PBS, fixed with 3.7% formaldehyde in PBS for 20 min, pre-incubated in blocking solution (PBS with 0.1% Tween-20% and 5% goat serum) and then incubated with primary antiserum diluted in blocking solution. The following primary antibodies were used: mouse anti-P1, mouse-anti-L1 (gifts from I. Ando), mouse anti-Hnt (AB_528278), mouse anti-Ptc (AB_528441), Rat anti-Shg (AB_528120), mouse anti-Antp (AB_528082), mouse anti-Dlp (AB_528191) (Developmental Studies Hybridoma Bank), mouse anti-Col (gift from M. Crozatier) (Pennetier et al., 2012 (link)), mouse anti-β-galactosidase (Sigma), rat anti-Jumu, rabbit anti-dMyc (Santa Cruz), and mouse anti-P-Mad (gift from Ed Laufer, USA). Alexa Fluor 488-, Alexa Fluor 568- and Alexa Fluor 594-conjugated secondary antibodies (Molecular Probes) were used. ROS detection was performed as previously described (Evans et al., 2014 (link)) using dihydroethidium (Invitrogen). Images were obtained using a Zeiss LSM510 confocal microscope or a Zeiss Axioplan 2 microscope equipped with fluorescence optics. All staining was performed in samples from at least three independent experiments.

Imaginal discs were dissected and fixed according to standard protocols. Primary antibodies used were guinea-pig anti-Hth [74 (link)], rabbit anti-PH3 (Sigma), rabbit anti β-galactosidase (Cappel), mouse anti β-galactosidase (Sigma), mouse anti-CD2 (Serotec), rabbit anti-GFP (Molecular Probes), mouse anti-Ey (Clements et al., 2008) and rabbit anti-cyclin B [75 (link)]. Mouse anti-Eya, rat anti-ELAV (7E8A10), and mouse anti-cyclin B were from Developmental Studies Hybridoma Bank (Iowa University). Fluorescently labeled secondary antibodies were from Molecular Probes. Anti-mouse-HRP (Sigma) was used for immunoperoxidase staining. Digoxigenin labelled stg RNA probe was produced from cDNA clone LD47579 (BDGP). ImageJ was used to quantify pixel intensities (http://imagej.nih.gov/ij/).

Staining of larval tissues was performed as described previously^{38 (link),44 (link)}. Larvae were dissected in PBS, fixed with 4% formaldehyde for 40 minutes on ice and then permeabilized for 15 minutes at room temperature in PBS containing 0.5% NP-40. The following primary antibodies were used in overnight incubations at 4 °C in blocking solution: rabbit anti-Apt (1:1000), rabbit anti-β-galactosidase (1:2000, Cappel), rabbit Caspase3 (1:50, Cell Signaling Technology), mouse anti-β-galactosidase (1:500, Sigma), mouse anti-Ubx (1:10, Developmental Studies Hybridoma Bank (DSHB)), goat anti-Cyclin E (1:200, Santa Cruz). The secondary antibodies used were as follows: Alexa 488 donkey anti-rabbit IgG conjugate (1:500, Molecular Probes), Alexa 488 donkey anti-mouse IgG (1:500, Molecular Probes), Cy3-conjugated donkey anti-mouse IgG (1:500, Sigma), Cy3-conjugated goat anti-rabbit IgG (1:500, CWBIO), bovine anti-goat IgG-CFL 555 (1:500, Santa Cruz). Mounting used VECTASHIELD Mounting Medium with DAPI (Vector Labs). The caspase-3 staining was did as described previously^{45 (link)}.

3^rd instar larval eye imaginal discs were dissected in PBS and fixed for 17 minutes in 4% formaldehyde, followed by three 10-min washes in PBS supplemented with 0.3% Triton-X-100 (PBT) and 30-min blocking in PBT containing 5% normal donkey serum (NDS). After blocking, discs were incubated with primary antibody either overnight at 4°C or 2 hours at room temperature in PBT containing 5% NDS. After incubation with primary antibody, discs were washed three times in PBT before incubating with secondary antibody in PBT containing 5% NDS for one hour at room temperature. After three subsequent washes, discs were mounted with glycerol. Primary antibodies used in this study include mouse anti-H2AK118ub (1:100, Millipore, E6C5), rabbit anti-H3K27me3 (1:100, Millipore), rabbit anti-H3K4me1 (1:100, Active Motif), rabbit anti-H3S10ph (1:500, Millipore), mouse anti-Ubx (1:20, DSHB, Ubx), mouse anti-Antp (1:20, DSHB, 8C11), rabbit anti-upd3 (1:750), and mouse anti-β-Galactosidase (1:1000, Sigma, GAL-50). Secondary antibodies include goat anti-mouse Cy3 (1:1000, Jackson ImmunoResearch), goat anti-mouse Cy5 (1:1000, Jackson ImmunoResearch), and goat anti-rabbit Cy3 (1:1000, Jackson ImmunoResearch).

Immunostainings of fly imaginal discs was performed by standard protocol. The following antibodies were used in this study: rabbit anti-β-galactosidase (Cappel), mouse anti-β-galactosidase (Sigma-Aldrich, #G4644), guinea pig anti-Dll [9 (link)], rat anti-Sp1, guinea pig anti-Hth [54 (link)], mouse-anti-GFP (ThermoFisher Scientific, #A11121), rat anti-C15 [15 (link)], rat anti-Al [6 (link)], rat anti-BarH1 [55 (link)], rabbit anti-BarH1 [56 (link)], mouse anti-Dac [57 (link)]. AlexaFluor488-, AlexaFluor555-, and AlexaFluor647-conjugated secondary antibodies from ThermoFisher Scientific or Jackson ImmunoResearch Laboratories were used at 1⫶500 dilution.
Adult legs were dissected, mounted, and analyzed by light microscopy. All adults of the relevant genotype that eclosed within an 8-hour period were scored. Roman numerals in the figure legends indicate the tarsal segments present in each phenotypic class (with the distal most segment perturbed). For example, a truncation designated as I-III means that tarsal segments I, II and III were present, with segment III partially defective (e.g. Fig 2P). n refers to the number of individual legs scored. The number of legs examined for each genotype is reported in the figures and figure legends.