Immunostaining of Murine Papillomavirus

Skin necropsies were snap frozen in Tissue-Tek OCT Compound freezing medium (Sakura Finetek USA Inc.) and HE- and IFS performed on ethanol-fixed tissue sections of 6 µm thickness [9] (link), [13] (link). For detection of MusPV1 L1 protein, sections were stained with a rabbit polyclonal immune serum directed against MusPV1 L1 at a dilution of 1∶4000 and detected with either an Alexa Fluor 488 or an Alexa Fluor 594-conjugated donkey anti-rabbit secondary antibody (both Life Technologies), as indicated. Co-stainings with either directly conjugated Alexa Fluor 488-anti-mouse CD4 or Alexa Fluor 488-anti-mouse CD8a antibodies (both Biolegend; dilution 1∶100) were performed to detect CD4⁺ or CD8⁺ T cells, respectively. To determine localization of MusPV1 L1 in relation to basal keratinocytes, sections were co-stained with a phycoerythrin-conjugated anti-CD49f antibody (integrin alpha 6, BD Biosciences). Nuclei were visualized by mounting sections with ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (LifeTechnologies). All microscopy analyses were performed on a Zeiss LSM 510 UV system and color levels of images were processed equally in Adobe Photoshop across experiments.