Chick anti gfp

Immunostaining for cultured cells was performed on cover glasses coated with human fibronectin. Cells was fixed by 4%PFA on ice for 10 min, then blocked using 3% skim milk, followed by incubating with primary antibodies; mouse-anti-FLAG(M2) (1/5000, Sigma), guinea pig anti-Ripply2 (1/100) (Zhao et al., 2015 (link)), and chick anti-GFP (1/400, Aves Labs, Oregon, USA) at 4°C overnight and then incubated with secondary antibodies; Alexa Fluor 488-conjugated anti-rabbit antibody (1/800, Life Technologies, Oregon, USA), Alexa Fluor 594-conjugated anti-mouse antibody (1/800, Life Technologies), Cy5 conjugated anti-Guinea pig antibody. The methods used for whole mount immunostaining and section immunostaining are described in our previous reports (Zhao et al., 2015 (link)). Antibodies used in this study, anti-Tbx6 and anti-Ripply2, were described previously (White and Chapman, 2005 (link); Zhao et al., 2015 (link)). For immunostaining of heart sections, we used rabbit-anti-TBX5 (H-70)(1/300, Santa Cruz Biotechnology, CA, USA), goat-anti-TBX18 (C-20) (1/300, Santa Cruz Biotechnology) as first antibodies. These sections were observed using FluoView FV1200 laser scanning confocal microscopy (Olympus).

Postnatal day 0 (P0) pups were injected with 60 mg/kg pimonidazole (Hypoxyprobe^TM, Burlington, MA, USA) in sterile PBS; mice were sacrificed after 90 min and kidney tissue was immediately collected and fixed in 4% PFA overnight at 4 °C. Staining was performed with 1:50 dilution used for the pimonidazole-specific primary and FITC secondary antibodies; chick anti-GFP (1:250; Aves Labs) and guinea pig anti-cytokeratin 8 (1:250; Abcam, Cambridge, MA, USA, Catalog #ab194130) were used as described above with secondary antibodies as indicated (Alexa Fluor 647 goat anti-guinea pig; 1:250; ThermoFisher Scientific, Catalog #A-21450; and Cy3 donkey anti-chicken IgY; 1:250; Jackson ImmunoResearch, Catalog # 703-165-155). Quantification in GFP+ regions normalized to signal in adjacent tubules was performed in ImageJ.