Mouse anti delta

Guts were dissected in PBS, fixed for 45 min at room temperature in 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO₄, 4 mM sodium phosphate, 1 mM MgCl₂, and 4%formaldehyde, washed for 1 hr, and incubated with primary antibodies and second antibodies in washing buffer (PBS, 0.5% BSA, 0.1% Triton X-100).
The following primary antibodies were used:, guinea-pig anti-hsc3 antibody antibody [60] (link) (1∶150), mouse anti-Delta (Developmental Studies Hybridoma Bank, 1∶100), rat anti-Delta (gift from Dr. MD Rand, University of Rochester, 1∶1000); rabbit anti-PH3 (phosphorylated histone H3, Upstate, 1∶1000), mouse anti-β-galactosidase (Developmental Studies Hybridoma Bank, 1∶500), rabbit anti-β-galactosidase (Cappel, 1∶5000), rabbit anti-peIF2α antibody (Cell Signaling: 3597, 1∶150), mouse anti-pJNK antibody (Cell Signaling: 9255,1∶150).
For Delta antibody staining, guts were fixed using a methanol-heptane method as descried [61] (link).
Fluorescent secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. DNA was stained using DAPI. Confocal imaging was performed on a Zeiss LSM700 confocal microscope and processed using ImageJ and Adobe Illustrator.

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).