Immunofluorescence Analysis of Hedgehog Signaling

Samples were prepared for immunofluorescence as published previously.^{4 ,5 (link)} Samples were fixed with 4% paraformaldehyde and permeabilized before 1-h blocking using 1 × PBS containing 10% horse serum. Afterward, samples were incubated overnight at 4°C with primary antibodies: polyclonal rabbit anti-Shh (1:100; Abcam) and sheep anti-CDON (1:100; R&D Systems). After three 5 min PBS-Tween20 (0.05%) rinses, secondary antibodies were incubated for 1 h (1:200) followed by Draq-5 (1:1,000; Thermo Fisher Scientific) stain. Samples were rinsed before mounted using Vectashield (Vector Laboratories). Images were collected on an upright microscope (DM5000 B; Leica) coupled to a confocal laser scanner (TCS SP5; Leica). LAS AF SP5 software (Leica) was used for data acquisition and analysis.