For HPV infection studies, cells were fixed with 4% paraformaldehyde (freshly made) for 25 min followed by 3 × wash step with PBS (Life technologies, 14190-169). After permeabilization in 0.3% Triton X-100 (Sigma, X100-500 ml), cells were washed again 3 × with PBS before staining with DAPI (1 µg/ml) for 15 min. Plates were maintained in PBS-NaN3 at 4 °C pending microscopic imaging. For IF, cells were blocked in 50% FBS after the permeabilization for 30 min and primary antibodies were added for one h. Following primary antibodies were used: mouse anti-lamin A/C (Santa Cruz Biotechnology, sc-376248, 1/100), rabbit anti-lamin B1 (Abcam, ab16048, 1/250), rabbit anti-lamin B2 (Abcam ab151735, 1/500), rabbit anti-PML (Santa Cruz, sc5621, 1/250), mouse anti-H3K9me2,3 (Cell Signaling Technology, #5327, 1/100), rabbit anti-H3K9ac (Cell Signaling Technology, #9649, 1/400), mouse anti-IRF3 (Abcam, ab68481, 1/100) and rabbit anti-p65 (Abcam, ab7970, 5 µg/ml). After 3 × 5 min wash step with PBS, the secondary antibodies; donkey anti-mouse CY3 (Jackson, 715-165-151, 1/600) and donkey anti-rabbit CY5 (Jackson, 711-175-152, 1/600), were added for 30 min. Afterwards cells were washed again 3 × 5 min with PBS and stained with DAPI similar as above. For transcription factor localization experiments additional HCS CellMask™ staining (Life Technologies, H32721, 1/5000) was applied, to allow distinguishing nuclear from cellular signal. To visualize EdU-labeled pseudogenomes, we made use of the Click-iT EdU Alexa Fluor™ 555 imaging kit (Invitrogen, C10338). Cells were incubated for 30 min at room temperature with the Click-iT reagent after blocking. Anonymized archival paraffin embedded human cervix samples, were microtome sectioned onto SuperFrost slides, deparaffinized (xylene) and rehydrated. After antigen retrieval (citrate buffer), they were subjected to an immunostaining for lamin A/C, lamin B1 and/or BAF (Abcam, ab129184, 1/500), counterstained with DAPI and mounted with Citifluor.
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