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).