A 21045

Quail embryos were fixed in 4% paraformaldehyde, embedded in 7% agarose, and vibratome sectioned at 100-μm thickness. The quail monoclonal endothelial cell surface antibody (1:50, QH1, Developmental Studies Hybridoma Bank, RRID:AB_531829, see [33 (link)]) and chick neural crest membrane marker HNK1 (1:500, TIB-200 hybridoma cell line, ATCC Cell Lines, RRID: AB_10013722, see [34 (link)]) were used to stain the tissue overnight at 4 °C. Secondary antibodies, goat anti-mouse, either Alexa Fluor 546 or 488 for QH1 and HNK1, respectively (1:500, A-21045 RRID: AB_10013722, and A-11030 RRID: AB_2534089, Thermo Fisher), were incubated for 2 h at ambient temperature. Stained sections were imaged by confocal microscopy (Zeiss, LSM 710).

NE-treated cells were fixed for 15 min using 4% paraformaldehyde and then blocked for 30 min using 5% donkey serum containing 0.3% Triton X-100. The cells were then incubated with the primary antibodies against CHGB (1 : 100; rabbit; PA5-52605, ThermoFisher Scientific) or SYP (1 : 100; rabbit; MA5-14532, ThermoFisher Scientific) for 3 and 1 h, respectively, followed by incubation with anti-rabbit Alexa Fluor® 488 Conjugate (1 : 100, A11034, ThermoFisher Scientific) secondary antibody for 1 h at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.
For Adrβ2 and cytokeratin expression in tumor and adjacent normal tissues, the tissues were incubated with primary antibodies against Adrβ2 (1 : 100; rabbit; PA5-14117, ThermoFisher Scientific) and cytokeratin (1 : 100; mouse; MA1-82041; ThermoFisher Scientific) for 1 h at room temperature, followed by incubation with a cocktail of anti-rabbit Alexa Fluor® 488 Conjugate (1 : 100, A11034, ThermoFisher Scientific) and anti-mouse Alexa Fluor® 546 (1 : 100, A21045, ThermoFisher Scientific) for 1 h at room temperature. Cells were then mounted using slow-fade DAPI (S36973, Life Technologies) and examined using Axio Observer 7.