FIG. 4.

Immunohistochemical analyses of the differentiation program in organotypic raft cultures. Shown are organotypic raft cultures of HPV16^WT-harboring NIKS cells (A, D, and G), HPV16^E5XCM−-harboring NIKS cells (B, E, and H), and untransfected NIKS cells (C, F, and I) that were maintained on a dermal equivalent of collagen embedded with fibroblasts. The cultures were lifted to the liquid-air interface after 4 days in culture and were harvested 11 days postlift. The rafts were fixed in 4% formalin, embedded in paraffin, and cut into 4-μm-thick serial sections. Cross sections from each sample stained with hematoxylin and eosin (A to C) reveal normal stratification of the keratinocyte cultures, with no gross morphological differences between the HPV16^WT (A)- and HPV16^E5XCM− (B)-harboring rafts. Immunohistochemical staining for terminal differentiation markers of the epithelium reveals that there is also no difference in the differentiation program between HPV16^WT- and HPV16^E5XCM−-harboring rafts. K10 was detected by immunohistochemical staining using an anti-K10 antibody (clone Ck 8.60). (D to F) Positive cells, staining brown, were localized to the suprabasal compartment of the epithelium, as expected. (G to I) Filaggrin was detected with an antifilaggrin antibody. Positive cells localized to the granular layer of the rafts, as expected.