Tsa cyanine 3 system

Embryos were dechorionated, fixed, immunostained and flat preparations performed according to published protocols (Patel, 1994 (link); Jussen and Urbach, 2014 (link)). The antibodies used are detailed in the supplementary Materials and Methods. Probes for in situ hybridization were synthesized using the templates shown in Table S4. In situ hybridization was performed as described previously (Jussen and Urbach, 2014 (link)) and probes processed with NBT/BCIP solution for non-fluorescent staining (Carl Roth) or tyramide signal amplification (TSA Cyanine 3 System; PerkinElmer) for fluorescent stainings. Embryos were then immunolabeled with primary antibody followed by incubation with biotinylated (processed with DAB) or fluorescent dye-coupled secondary antibodies as described in the supplementary Materials and Methods. Non-fluorescent stainings were documented on a Zeiss Axioplan microscope, while fluorescent confocal images were acquired on a Leica TCS SP5 II microscope. Images were processed with ImageJ (NIH), Adobe Photoshop and Adobe Illustrator.

Prior to double immunohistochemistry, E10.5 tails were fixed, sectioned to generate sagittal paraffin sections (7 µm) and antigen was retrieved. Endogenous horseradish peroxidase (HRP) activity in the sections was quenched using 1% hydrogen peroxide. Primary antibody incubation for Notch1 (purified mouse anti-mouse Notch1; clone mN1A Cell Signaling Technology; 1:20), Dll1 [rat anti-Dll1 monoclonal antibody (mAb); PGPM-1F9; kindly provided by E. Kremmer (Geffers et al., 2007 (link)); 1:50] and NICD [cleaved Notch1 (Val1744); D3B8; rabbit mAb Cell Signaling Technology; 1:200] was conducted in 10% NGS in PBST for 24-72 h at 4°C. Dll1 and Notch1 antibody binding was detected using Alexa Fluor-conjugated secondary antibodies, whereas NICD signal amplification and detection were performed using a TSA-Cyanine 3 system (PerkinElmer). Samples were co-stained with DAPI (1 mg/ml in PBS), mounted in Prolong Gold and imaged on a Zeiss 710 confocal microscope. We performed the following controls: no primary antibody, no primary or secondary antibodies, each antibody separately in single immunohistochemistry. See supplementary material Table S3 for details of antibodies.