Anti keratin 14

10-µm frozen tissue sections were rehydrated in 0.2% Tween 20/PBS for 10 min, fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton X-100, blocked in 10% heat-inactivated goat serum/5% milk/PBS for 1 h, and then stained with anti–cleaved caspase 3, anti-CD31 (BD), anti–LN-332 (Abcam), anti-FN (Sigma-Aldrich), anti–MMP-9 (Sigma-Aldrich), or anti–keratin 14 (Covance). Immunostaining was also performed using antibodies against integrin α2 (CD49b; EMD Millipore), α5 (5H10-27; BD), and α6 (GoH3; EMD Millipore). Secondary antibodies were Alexa Fluor 488 goat anti–rat IgG, Alexa Fluor 488 goat anti–hamster IgG, Alexa Fluor 594 goat anti–mouse IgG, or Alexa Fluor 594 goat anti–rabbit IgG (Molecular Probes), as appropriate. Images were collected on an Eclipse 80i upright microscope using a Spot camera. For assessment of wound immunostaining, the field within the wound bed below the reepithelialized epidermis was imaged. For assessment of vessel density within papillomas, nonnecrotic tumor regions were imaged. CD31 and MMP-9 staining was quantified using ImageJ software.

4-µm paraffin sections were deparaffinized and rehydrated. For antigen retrieval, the slides were autoclaved in 10 mM sodium citrate buffer. Sections were blocked in PBS containing 10% BSA at room temperature for 30 min. The sections were incubated overnight at 4°C with the following dilutions of primary antibodies: anti–β-catenin (1:100; BD), anti-CXXC5 (1:50; Santa Cruz Biotechnology, Inc.), anti–keratin 14 (1:1,000; Covance), anti–collagen I (1:500; Abcam), anti-PCNA (1:500; Santa Cruz Biotechnology, Inc.), anti-vimentin (1:250; Abcam), anti-CD34 (1:500; Abcam), anti-CD68 (1:200; Abcam), and anti–p-Erk (1:50; Cell signaling Technology). The slides were washed with PBS, incubated with Alexa Fluor 488– or Alexa Fluor 555–conjugated IgG secondary antibody (1:400; Molecular Probes) at room temperature for 1 h, and counterstained with DAPI (1:5,000; Boehringer Mannheim). The images were captured using a LSM510 META confocal microscope (Carl Zeiss) after excitation with 405-, 488-, or 543-nm laser lines.