Mouse anti antp

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.

The dome-Gal4 line was crossed with appropriate hhF4f-GFP;UAS-RNAi lines, mid-third instar larvae were collected and lymph glands were dissected lymph glands. Immunostaining was performed as described previously [14 (link)]. The following antibodies were used to identify PSC cells: mouse anti-Antp (primary antibody; 1:100; 4C3, Developmental Studies Hybridoma Bank); Alexa 555-conjugated mouse IgG antibody (secondary antibody; A28180, Thermo Fisher Scientific). Cell nuclei were stained with DAPI (Invitrogen). Immunostained samples were analysed with a Nikon A1R laser-scanning confocal microscope.