Histological and Epigenetic Analysis of Oral Lichen Planus

We reassess the histopathological slides of the 42 patients that received diagnosis of OLP based on the criteria proposed by the WHO.^{22 (link)} Histological signs of dysplasia were exclusion criteria. Immunofluorescence was performed as previously reported^{5 (link)} using a double staining with anti acetyl-histone H3 (cell signaling) and vimentin (DAKO, Carpinteria, CA) and anti phospho-histone H2A.X (Millipore, Billerica, CA) as primary antibodies followed by FITC or TRITC-conjugated secondary antibody (Covance, Berkeley, CA). DNA was stained using Hoechst 33342. Images of 4 to 10 fields of each case were captured at 400× magnification using a QImaging-ExiAqua monochrome digital camera attached to a Nikon Microscope (Nikon, Melville, NY) and visualized with QCapturePro 7 software (Surrey, BC, Canada). We performed morphometric image analysis for γH2AX using the software ImageJ (Version 1.38s; NIH, Bethesda, MD). All positive and negative cells were counted in each field and the percentage of total number of cells in each case was calculated. For ac.H3K9ac staining intensity analysis, all cases were classified as negative (0), weak (+), moderate (++), or strong (+++). The intensity of H3K9ac was graduated independently by 3 oral pathologists.