Figure 4

Distribution of the viral and cellular markers E4, L1, human papillomavirus 25 (HPV25) DNA and minichromosome maintenance protein 7 (MCM7) in three cases of hypertrophic actinic keratosis from a kidney transplant recipient (patient 16, a. hand; b. face; c. hand). (a) The top picture (scale bar: 1000 μm) shows the scan of the tissue section of the first actinic keratosis using hematoxylin and eosin (H&E) staining. The region shown in the lower panels corresponds to the red square highlighted in the over H&E image, reproduced in the lower left-hand picture. The same section was double stained using antibodies to E4 (green) and MCM (red) (second picture); serial sections were double stained for the presence of viral genome amplification by HPV25 DNA-fluorescent in situ hybridization (FISH) (red) and for E4 expression (green) (third picture), and also stained with antibodies to L1 (red) (fourth picture). The white arrows indicate nuclear L1 staining. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) to visualize cell nuclei. Scale bars: 50 μm. (b) The top pictures (scale bars: 1000 μm) show the scan of the tissue sections (using H&E staining) of the second actinic keratosis (left picture) and its edge (right picture). The right-handed middle panel shows the histology (H&E staining) of the area from the edge where viral markers were expressed (red square) and an overall picture of the immunofluorescence (IF) staining (E4 in green; MCM7 in red) of the same tissue section (scale bars: 200 μm). The white dotted line indicates the basal layer. In the lower panel, the above MCM7/E4 staining is reproduced (left picture) and serial sections were stained with antibodies to L1 (red) (right picture). The white arrows indicate nuclear L1 staining. Scale bars: 100 μm. All sections were counterstained with DAPI (blue) to visualize cell nuclei. (c) The top picture shows the scan of the tissue section of the third actinic keratosis using H&E staining (scale bar: 1000 μm). The black rectangle shows a magnification (scale bar: 400 μm) of the positive parakeratotic epithelium that abruptly changed to normal orthokeratosis on both sides. The region shown in the lower panels corresponds to the red square highlighted in the H&E image, reproduced in the lower left-hand picture. The same section was double stained using antibodies to E4 (green) and MCM7 (red) (middle picture) and a serial section was stained with antibodies to L1 (red) (right picture). Scale bars: 50 μm. All sections were counterstained with DAPI (blue) to visualize cell nuclei.