Rabbit anti dmyc

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.

Fluorescent staining was performed using the following antibodies: rabbit anti-Twi (Figeac et al., 2010 (link)) (1:300), rabbit anti-dMyc (1:300) (Santa-Cruz Biotechnology), goat anti-GFP (1:1000) (Biogenesis), rabbit anti-PH3 (1:1000) (Millipore), mouse anti-NICD (1:150), rat anti-Deltex (1:50) (kindly provided by S. Artavanis-Tsakonas, Harvard Medical School, USA), mouse anti-Lamin (1:1000) (DHSB LC28.26), rat anti-Tropomyosin (1–200; Babraham Bioscience Technologies, UK; BT-GB-141), mouse anti-αPS1 (1:50; DHSB; DK.1A4), mouse anti-βPS (DSHB CF.6G11), Phalloidin-TRITC (1:1000) (Sigma). Cy3, Cy5 and Alexa 488-conjugated secondary antibodies (Jackson ImmunoResearch) were used (1:300). Embryos were mounted in Fluoromount-G anti-fade reagent (Southern Biotech). Labeled embryos were analyzed using Leica SP5 and SP8 confocal microscopes. 3D reconstructions of the images were generated using Imaris software (Bitplane).