Paraffin sections were deparaffinized in xylene, rehydrated in a decreased ethanol series, and subjected to antigen retrieval in 10 mM sodium citrate buffer by boiling for 10 minutes.
21,22 Sections were treated with 3% hydrogen peroxide in PBS for 20 minutes to block endogenous peroxidase activity. Cyrosections (7 μm) were dipped in PBS to remove OCT, fixed with 4% PFA for 10 minutes, and then rinsed with PBS three times. Tissue sections were blocked with 3% horse serum in PBST (PBS plus 0.1% Tween-20 for paraffin sections) or PBS plus 0.5% Triton X-100 (for cryosections) for 1 hour at room temperature and then incubated overnight at 4°C, with primary antibodies diluted in the same buffer. Slides were washed with PBS, incubated at room temperature for 1 hour with either fluorophore-conjugated or biotinylated secondary antibodies, and then washed with PBS. For immunofluorescence, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and slides were mounted with Mowiol (Sanofi-Aventis, Bridgewater, NJ, USA) for viewing under a Leica microscope equipped with a charge-coupled device (CCD) camera for photography. For immunohistochemistry, sections were incubated with Vectastain Elite ABC Reagent (PK-6100; Vector Laboratories, Burlingame, CA, USA), and color was developed by using 3,3′-diaminobenzidine as a substrate (D4293; Sigma-Aldrich Corp., St Louis, MO, USA). Sections were counterstained with hematoxylin for cell nuclei.
Primary antibodies were purchased from the following sources: anti-FGFR2 (ab10648; Abcam, Cambridge, MA, USA); anti-keratin 10 and anti-keratin 14 (PRB-159P and PRB-155P, respectively; Covance, Emeryville, CA, USA); anti-keratin 16 (NBP2-45538; Novusbio, Littleton, CO, USA); anti-p63 (GTX102425, GeneTex, Irvine, CA, USA), anti-PPARγ (#2435; Cell Signaling, Danvers, MA, USA); anti-PCNA (ab29; Abcam); and anti-BrdU (M-0744; Dako, Carpinteria, CA, USA). Fluorophore-conjugated secondary antibodies were obtained from ThermoFisher Scientific (Rockford, IL, USA) and biotinylated secondary antibodies were from Vector Laboratories.