Mouse anti histone

Tissues were dissected 72 hAH in 3.7% formaldehyde and fixed for 25 minutes at RT. Tissues were washed in 0.4% Triton X-100 in 1X PBS (0.4% PBT), 3X 15 minutes, and blocked in 10% NGS in 0.4% PBT at RT for 1–2 hours. Tissues were incubated in primary antibody in 0.4% PBT at 4°C overnight [1∶100 rat-anti Sima (P. Wappner), 1∶100 rabbit-anti phospho-Mad (E. De Robertis), 1∶100 mouse-anti Histone (Millipore)], then washed extensively in 0.4% PBT and incubated in secondary antibody in 10% NGS +0.4% PBT at 4°C overnight or 2 hours RT [mouse-FITC, rat-FITC, rabbit-Cy3 (Jackson Immunochemicals, PA)]. Tissues were again washed, incubated in 1∶500 TO-PRO3 (TOPRO; Invitrogen, CA) in distilled water for 30–60 minutes at RT (if noted), and mounted in Vectashield (Vector Laboratories, MI). Images were obtained using a Zeiss LSM5 confocal laser scanning microscope.
The procedure for Dilp2 analysis was adapted from [20] (link), and is the same as outlined above but with the following exceptions: 5% BSA in 0.4% PBT was used instead of 10% NGS in 0.4% PBT. Primary antibody was 1∶100 rat-anti Dilp2 (P. Leopold) and secondary antibody was rat-Cy3 (Jackson Immunochemicals, PA).

Flies were housed at 25°C under an LD cycle on standard media, unless otherwise noted. At each time point ∼10 intestines from female flies <14 days were dissected in PBS (Fisher) and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS and then counterstained with DAPI (Thermo Fisher Scientific, 1:5,000) in PBS-T (PBS + 0.2% Triton X-100, Fisher). Intestines were then blocked in 1% BSA (Bio Basic) + 0.2%Triton X-100 (Fisher) and incubated in the same at room temperature for 2 hr with primary antibodies: mouse anti-Delta (Developmental Studies Hybridoma Bank [DSHB], 1:50), mouse anti-prospero (DSHB, 1:50), mouse anti-histone (Millipore, 1:2000), or rabbit anti-PER (generously provided by Patrick Emery, 1:1,500), then incubated at room temperature for 1 hr in secondary goat anti-mouse/rabbit antibodies (Life Technologies, 1:2000), and counterstained with DAPI (Thermo Fisher Scientific, 1:5,000). Samples were imaged using a slide scanner (Zeiss Axio Scan.Z1) that assembled single images consisting of merged and tiled z stacks of the entire tissue sample in a single plane of focus, or by confocal microscopy (Olympus IX81 FV1000) with a 60× water-immersion lens. Images were analyzed using Zen Blue Edition software (Zeiss) and processed using Photoshop CS5 (Adobe).