Ta180004

Embryos were fixed in 4% paraformaldehyde overnight, then rinsed with PBST four times every 5 min. Next, the resulting embryos were blocked at room temperature for at least 1 h in PBST containing 10% heat-inactivated goat serum and 1% BSA, and then stained using the following affinity-purified primary antibodies overnight at 4°C: anti-ZsYellow (1:200; 632475, Clontech), anti-ZsYellow (1:800; TA180004, Origene), anti-Cdh5 (1:200; 555289, BD Pharmingen), anti-pERK1/2 (1:1000; 9101, Cell Signaling), anti-pAKT (1:400; 4060, Cell Signaling Technology), anti-Scl (1:200; NBP2-50285, Novus Biologicals), anti-Etv2 (1:500; ES1004, Kerafast), anti-GFP (1:1000; A11120, Invitrogen) and anti-Flag (1:500; M20008, Abmart). Samples were then washed for 3-4 h with PBST, followed by incubation with secondary antibodies for 1 h at room temperature, including DyLight 488-conjugated goat anti-rabbit IgG (1:200; 711-545-152, Jackson), DyLight 594-conjugated goat anti-mouse IgG (1:200; 715-585-150, Jackson), DyLight 488-conjugated AffiniPure goat anti-mouse IgG (1:200; 715-545-150, Jackson) and DyLight 594-conjugated AffiniPure goat anti-rabbit IgG (1:200; 711-585-152, Jackson). DAPI (1:10,000, Sigma) was used as a nuclear stain.

Immunofluorescence staining was performed as previously described (Ning et al., 2013 (link)). Embryos were stained with the following affinity-purified antibodies: anti-GFP (1:1000; A111201, Invitrogen), anti-ZsYellow (1:200; 632475, Clontech), anti-ZsYellow (1:400; TA180004, Origene) and anti-p-Smad1/5/8 (1:200; 9511, Cell Signaling Technology). Fluorescence in situ hybridization was performed as previously described (Schoenebeck et al., 2007 (link)). Anti-DIG HRP-conjugated Fab fragments (1:400; Roche) were used to detect the digoxigenin (DIG)-labeled probes. Embryos were then incubated with fluorescein (FLU) tyramide (1:100; PerkinElmer) for 3 h at 28.5°C. Next, the embryos were subjected to immunofluorescence after removal of HRP activity.

Digoxigenin-UTP-labeled antisense RNA probes were transcribed using MEGAscript Kit (Ambion) according to the manufacturer's instructions. Whole-mount in situ hybridizations were performed according to previously published methods (Ning et al., 2013 (link)). In particular, fluorescent in situ hybridization in immunostained embryos was conducted as previously described (Mao et al., 2021 (link)). Briefly, for the detection of cxcr4a and tie1 expression, Tg(nkx2.5:ZsYellow) embryos were first immunostained using affinity-purified anti-ZsYellow antibody (1:800; TA180004, Origene), and then subjected to in situ hybridization with digoxigenin-labeled cxcr4a or tie1 probe. Anti-digoxigenin-HRP (1:400; Roche, 11633716001) was used as primary antibody to detect the probes and the hybridization signals were visualized by incubating embryos with fluorescein tyramide (1:50; PerkinElmer, NEL701A001KT). For the detection of cxcl12b expression, Tg(sox17:GFP) embryos were fluorescently stained with anti-GFP (1:1000; A11120, Invitrogen) antibody. The resulting hybridization signals were similarly generated with cyanine 3 tyramide (1:50; PerkinElmer, NEL701A001KT).