Keratinocytes containing wild type HPV18 genomes or the mutant E6ΔPDZ genome were grown in organotypic raft cultures by seeding the keratinocytes onto collagen beds containing J2-3T3 fibroblasts [13 (link)]. Once confluent the collagen beds were transferred onto metal grids and fed from below with FCS-containing E media without EGF. The cells were allowed to stratify for 14 days before fixing with 4% formaldehyde. The rafts were paraffin-embedded and 4 μm tissue sections prepared (Propath UK, Ltd., Hereford, UK).
For analysis of Phospho-STAT3 (S727) (ab32143, abcam), Total STAT3 (C-20: sc-482, Santa Cruz Biotechnology and 9132, CST), involucrin (SY5, Santa Cruz Biotechnology) and HPV18 E1^E4 (mouse monoclonal antibody 1D11 [84 ]) expression, the formaldehyde-fixed raft sections were treated with the sodium citrate method of antigen retrieval. Briefly, sections were boiled in 10 mM sodium citrate with 0.05% Tween-20 for 10 minutes. Sections were incubated with appropriate antibodies and immune complexes visualized by using Alexa 488 and 594 secondary antibodies (Invitrogen). The nuclei were counterstained with the DNA stain 4’,6-diamidino-2-phenylindole (DAPI) and mounted in Prolong Gold (Invitrogen).
For analysis of Phospho-STAT3 (S727) (ab32143, abcam), Total STAT3 (C-20: sc-482, Santa Cruz Biotechnology and 9132, CST), involucrin (SY5, Santa Cruz Biotechnology) and HPV18 E1^E4 (mouse monoclonal antibody 1D11 [84 ]) expression, the formaldehyde-fixed raft sections were treated with the sodium citrate method of antigen retrieval. Briefly, sections were boiled in 10 mM sodium citrate with 0.05% Tween-20 for 10 minutes. Sections were incubated with appropriate antibodies and immune complexes visualized by using Alexa 488 and 594 secondary antibodies (Invitrogen). The nuclei were counterstained with the DNA stain 4’,6-diamidino-2-phenylindole (DAPI) and mounted in Prolong Gold (Invitrogen).
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