Eight well glass slides

Immunostaining was performed in paraffin sections according to the standard protocols. In brief, samples were deparaffinized and rehydrated, followed by antigen retrieval in 10 mM sodium citrate. Blocking and staining were performed in antibody diluent with background-reducing components (Dako). Sections were incubated with rat anti-BrdU (Abcam, #ab6326, 1:500) and rabbit anti-Pdgfrα (Cell Signaling Technology, #3174, 1:500) antibodies overnight at 4°C, followed by incubation with the corresponding secondary antibodies goat anti-rat Alexa Fluor 594 and goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, both used at 1:500), respectively. For HeLa cells overexpressing human wild-type or mutant β-catenin plasmids, cells were seeded in eight-well glass slides (Millipore). After transfection, cells were fixed in 4% PFA at 37°C for 30 min and then washed sequentially with PBS, 0.2% PBST (Phosphate buffered saline with Tween-20), and PBS. Blocking and staining proceeded as described above. Slides were mounted with DAPI (4′,6-diamidino-2-phenylindole) Fluoromount-G mounting media (Southern Biotech) and imaged by fluorescence microscopy (Olympus). Cell counting was achieved using the ImageJ software.