For routine histology, tissues were fixed in Bouin's fixative, processed, and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin and examined and photographed using an Axiophot microscope (Carl Zeiss, Inc.). For immunofluorescence, frozen sections of tissues or cells on the glass coverslips were fixed in 4% paraformaldehyde in PBS for 10 min and were subjected to indirect immunostaining (DasGupta and Fuchs 1999 ) and analyzed using a confocal microscope LSM 410 (Carl Zeiss, Inc.).
Unless otherwise indicated, primary antibodies were polyclonal and raised in rabbits. Antibodies and dilutions used were: rat monoclonal β1 (1:100), α3 (1:100), rat monoclonal α4 (1:50), rat monoclonal α6 (1:100) (Chemicon); K1 (1:200), loricrin (1:250), filaggrin (1:2000) (BabCo); laminin (1:200), mouse monoclonal Ki67 (1:100) (Sigma-Aldrich); K17 (1:1000; gift of P. Coulombe, Johns Hopkins University School of Medicine, Baltimore, MD); guinea pig polyclonal K5 (1:300); and Lef1 (1:250). Fluorescence-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories. DAPI was used to stain nuclei.
Unless otherwise indicated, primary antibodies were polyclonal and raised in rabbits. Antibodies and dilutions used were: rat monoclonal β1 (1:100), α3 (1:100), rat monoclonal α4 (1:50), rat monoclonal α6 (1:100) (Chemicon); K1 (1:200), loricrin (1:250), filaggrin (1:2000) (BabCo); laminin (1:200), mouse monoclonal Ki67 (1:100) (Sigma-Aldrich); K17 (1:1000; gift of P. Coulombe, Johns Hopkins University School of Medicine, Baltimore, MD); guinea pig polyclonal K5 (1:300); and Lef1 (1:250). Fluorescence-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories. DAPI was used to stain nuclei.
