2.14 Immunohistochemistry (IHC)
Paraffin sections were deparaffinized and rehydrated. For antigen retrieval, the slides were autoclaved or microwaved for 15 minutes in 10 mM sodium citrate buffer. The samples were pre-incubated in PBS and then blocked with 5% BSA in PBS for 30 minutes at room temperature. The samples were incubated overnight at 4°C with the following primary antibodies: anti-β-catenin (1:200, BD Transduction or 1:100, Abcam), anti-K15 (1:200, Abcam), anti-PCNA (1:500, Santa Cruz Technology), anti-Ki67 (1:500, Abcam), anti-Fgf9 (1:500, Abcam) and anti-Cytokeratin 17 (1:500, Abcam). After washing with PBS, the slides were incubated with anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 555 conjugated secondary antibody (1:500, Molecular Probes) for 1 hour at room temperature and counterstained with DAPI (1:3000, Boehringer Mannheim) for 10 minutes. The images were taken using a LSM510 confocal microscope (Carl Zeiss).