Immunohistochemistry Embryo Staining

For immunohistochemistry embryos were fixed and stained according to standard procedures. Guinea pig anti-SIMU (30 (link)) and guinea pig anti-Drpr (32 (link)) were used at a 1:5000 and 1:100 concentrations, respectively. Rabbit anti-activated caspase 3 (Dcp-1) (Cell Signaling) and mouse anti-GFP (Roche) were used at 1:100 concentration. Rabbit anti-Crq antibody (1:500) is a gift from N. Franc. Rabbit anti-Srp antibody (1:100) is a gift from J. Casanova, K. Campbell and N. Martin. Rabbit anti-Peroxidasin antibody (1:2000) is a gift from Jiwon Shim. Fluorescent secondary antibodies (Cy3/and Cy5/Jackson ImmunoResearch; Alexa Fluor 488/Molecular Probes) were used at 1:200 dilutions. For TUNEL labeling embryos were re-fixed, washed and labeled with the In Situ Cell Death Detection kit (Roche) according to the manufacture instructions. Images were acquired on a confocal microscope Zeiss LSM 700 or on a Zeiss Axio Observer microscope equipped with an Apotome system using the AxioVision software. 75% Glycerol solution was used as the imaging medium.
Live imaging was carried out by dechorionating embryos (stage 15), mounting them under Halocarbon oil, injecting 2–3% egg volume of LysoTracker (Molecular Probes) as described in Ref. (33 (link)). Recording started 30 min following injection.