Immunohistochemistry (IHC)

For the 81 included skin biopsies, IHC was conducted on about 4µm-thick paraffin sections on heat fixed positively charged slides. Deparaffinization, rehydration and epitope exposure were performed with use of 0.01 M citrate buffer (pH 6.0) for 10 minutes in microwave. Activity of endogenous peroxidase was blocked by 3% hydrogen peroxide incubation for 10 min. All sections were rinsed with phosphate buffer saline (PBS) and incubated for one hour at room temperature with the primary antibodies directed against: monoclonal mouse anti-human Ki67; a cell proliferation marker; (Dako corporation product clone: MIB-1, isotype: IgG1, kappa, ready to use for immunohistochemical staining of formalin-fixed, paraffin-embedded tissues, code: IR626, corresponding to a1002bp Ki67 molecule), and monoclonal mouse anti-human CD31 antibody; a neoangiogenesis and stem cell marker; (Dako corporation product clone: JC70A species, IgG1, kappa, cell culture supernatant dialyzed against 0.05 mol/L Tris-HCl, pH 7.2, and containing 15 mmol/L NaN3, concentrate, dilution 1:100). Standard avidin-biotin-peroxidase technique was applied using diaminobenzidine (DAB, 5 minutes incubation) for visualization and hematoxylin for counterstaining (30 seconds). Appropriate negative controls, consisting of histologic sections processed without the addition of primary antibody, were prepared, along with a positive control sections prepared from tonsillar tissue for CD31, while the basal epidermal cells were considered as an internal positive control for Ki67.