Dx41 microscope

The 3D epidermal skin equivalent were fixed with 10% formaldehyde, embedded with paraffin, and prepared as 0.4 μm sections using a RM2255 Microtome (Leica, Wetzlar, Germany). Immunohistochemistry was performed, using the avidin-biotin complex technique with Universal VECTASTATIN ABC Kit (Vector Laboratories, Burlingame, CA, USA). Paraffin sections were de-paraffinized in xylene, hydrated through a decreasing ethanol concentration grades, and endogenous peroxidase activity was quenched using 0.3% hydrogen peroxide in PBS. Non-specific binding was eliminated by incubating with diluted normal blocking serum. Sections were then serially incubated with primary antiserum diluted in buffer for 1 h, diluted biotinylated secondary antibody solution for 40 min, ABC reagent for 30 min, and peroxidase substrate solution until the desired stain intensity developed. All steps were performed at room temperature. For immunohistochemistry, we used monoclonal antibodies for cytokeratin 14 from Abcam (Cambridge, UK) and P63 from CHEMICON International Inc. (Temecula, CA, USA), Filaggrin from Novocastra Laboratories Ltd. (Newcastle, UK) and E-Cadherin form Santa Cruz Bio-technology Inc. Each section was counterstained with hematoxylin, mounted and entire tissue area was examined under on Olympus DX41 microscope (CenterValley, PA, USA).