Mouse anti β gal

The wing imaginal discs and pupal retinas were dissected from late third instar larvae and twenty-four hours after puparium formation (APF) pupae, respectively, then fixed for 17 min in phosphate-buffered saline (PBS) with 4% paraformaldehyde and then blocked in PBS with 0.3% Triton X and 5% BSA (bovine serum albumin) for 30 min at room temperature and incubated with primary antibodies overnight at 4°C. After washed with PBS-Triton X three times, the wing discs were incubated with secondary antibody for 1 h at room temperature. The stained wing discs were then mounted on the slides with glycerol. Primary antibodies: mouse anti-Cut (2B10, 1:200, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA), mouse anti-Delta (C594.B9, 1:200, DSHB), mouse anti-Notch (C17.9C6, 1:100, DSHB), mouse anti-β-gal (1:1000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-cleaved caspase-3 (1:200; Abcam). Secondary antibodies [anti-mouse Cy3 (1:1000), anti- rabbit Cy5 (1:1000)] were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The fluorescent images were acquired by confocal microscope TCS SP5 (Imaging Core, First Core Labs, National Taiwan University College of Medicine) and Axio Imager A1 Microscope (Zeiss, Thornwood, NY, USA). Leica LAS AF Lite and Adobe Photoshop were used to analyze and process images.

Flies were transferred overnight into a classical fly food vial containing a filter paper soaked with a solution consisting of 5% sucrose to clean the digestive tract. Then, 10–15 intestines of mated adult females were dissected in phosphate-buffered saline (PBS), and fixed for at least one hour at room temperature in 4% paraformaldehyde (PFA) in PBS. They were subsequently rinsed in PBS+0.1% Triton X-100 (PBT), permeabilized and blocked in 2% BSA PBT for one hour, and incubated with primary antibodies in 2% BSA PBT for overnight at 4°C. After one hour of washing, secondary antibodies and DAPI were applied at room temperature for two hours.
Primary antibodies used are: mouse anti-Pros (DSHB, 1:100), mouse anti-Arm (DSHB, 1:100), mouse anti-Dl (DSHB, 1:100), mouse anti-βPS (DSHB, 1:100), mouse anti-Con (DSHB, 1:4), rabbit anti-pH3 (Millipore, 1:1000), Chicken anti-GFP (Abcam, 1:1000), rabbit anti-βGal (Cappel, 1:1000), mouse anti-βGal (Sigma, 1:1000), and Rat anti-mCherry (Life Technologies, 1:500). Alexa488-, Alexa555- or Alexa647-conjugated secondary antibodies (Life Technologies) were used. Nuclei were counterstained by DAPI (Sigma, 1:10’000). All the images were taken on a Zeiss LSM 700 confocal microscope at BIOP in EPFL. Images were processed using Image J and Adobe Photoshop software. Shown in figures are maximal intensity projections of all the confocal z stacks.