Tsa plus cyanine 3 system

Immunostaining was performed as previously described (Le Guen et al., 2014 (link); Shin et al., 2016 (link)), with the addition of a 45 min at room temperature digestion step in proteinase K (PK) as described previously (Koltowska et al., 2015b (link)). The primary antibodies used were anti-Prox1 rabbit (AngioBio, #11-002P, 1:100, Lot: GR3247830-10) (Koltowska et al., 2015a (link)) and anti-mCherry chicken (AvesLabs, #MCHERRY-0020, 1:100, Lot: MC87977980). For the anti-mCherry primary antibody, we verified that the observed signal recapitulated the endogenous transgenic line expression. The secondary antibodies used were anti-rabbit IgG HRP (Cell Signaling Technology, #7076, 1:1000, Lot: 28) and anti-chicken 488 (Jackson ImmunoResearch, #703-545-155, 1:200, Lot: 158347). Signal amplification was performed using the TSA™ Plus Cyanine 3 System (Perkin Elmer, #NEL744001KT), with a development time of 3 h. Imaging was performed as described above.

Embryos were treated with 15 μM of the chemical inhibitor SL327 (Merk, NJ, USA) diluted in E3 medium with 0.003% 1-phenyl-2-thiourea (PTU) and 1% DMSO (Sigma) and immobilised with Tricaine (0.08 mg/ml) and 1% low melting agarose for imaging. Control embryos were kept in E3-PTU water with 1% DMSO.
Immunofluorescent staining was performed as described in Okuda et al. (2018) (link) with the following minor changes to the protocol: 30 min of ProtK treatment (20 ng/ml) was used for 36 hpf old embryos. For EGFP, the primary antibody used was chicken a-GFP (1:400, Abcam, #ab13970) and secondary C1 anti-GFP (Invitrogen, #A11039). For Prox1, rabbit a-Prox1 (1:500, AngioBio, #11–002) was used as primary and a-rabbit IgG-HRP (1:1,000, Cell Signaling, #7074S) as secondary and amplified the Prox1 signal with TSA Plus Cyanine 3 System (Perkin Elmer, #NEL744001KT).

Embryonic day 3 (E3) and E4 chick embryos were fixed and embedded in gelatin (7.5% gelatin, 15% sucrose in PBS). 14μm thick sections were collected on Superfrost Plus slides. For AP2a and Jagged-1 co-detection, slides were boiled in 10mM citric acid for 10 minutes prior to antibody application and then incubated in 0.012% hydrogen peroxide for 15 minutes at room temperature. The 3B5 AP2α monoclonal antibody developed by Trevor Williams was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242. AP2α antibody was diluted 1:100 and Jagged-1 polyclonal antibody (Santa Cruz Biotechnology H-114) was diluted 1:200 in blocking buffer (PBS with 0.02% Tween-20, 0.1% Triton X-100, and 10% goat serum). Staining was detected with biotinylated mouse secondary antibody (Mouse Vectastain ABC kit) in conjunction with PerkinElmer TSA Plus Cyanine-3 System and AlexaFluor 488 conjugated rabbit secondary antibody diluted 1:500 in A+B substrate solution (AlexaFluor goat anti-rabbit, Invitrogen). All slides were mounted in Fluoromount G (Southern Biotech).