Once obtained, each sample was divided in three fragments, which were fixed, respectively, in 4.0% w/v neutral buffered formaldehyde (for light microscopy, histochemistry and immunohistochemistry for Melan-A, CD1A, Smoothelin, SMA-ACT, CD-31 and D2-40), methacarn fixative consisting of 60% methanol, 30% chloroform and 10% glacial acetic acid (for immunohistochemistry for CK5/6, CK7, CK10, CK20, claudin-1, DSPK 1-2, involucrin, filaggrin, laminin, collagen types I, III and IV, decorin, biglycan and versican) and 2.5% glutaraldehyde (for electron microscopy). Samples fixed in formaldehyde or methacarn were embedded in paraffin, and 5 µm tissue sections were obtained for hematoxylin and eosin (H&E) staining using routine methods. H&E stained sections were photographed using a Nikon Eclipse 90i light microscope (Nikon Corp., Tokyo, Japan) and thickness of each epithelial stratum was quantified in each sample using a scale as a reference. For this purpose, 30 equidistant vertical lines were drawn in each histological image using a predesigned template, and height of each cell layer was assessed at each point. This way, both the rete ridges and the papillae were measured and the average of all 30 measured was calculated.
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