Permanox lab tek chamber

Cells were seeded in 2-well Permanox Lab-Tek chamber slides (Nunc, Thermo Fisher) and infected at 80–90% confluency with WT BoHV-1, ΔgM BoHV-1, or ΔgM Rev BoHV-1 at various MOIs. Cells were fixed at different times post infection with 4% paraformaldehyde (Sigma-Aldrich Canada Ltd.), washed with phosphate buffered saline (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4 (PBS)), and then blocked overnight with 1% goat serum. The cells were permeabilized the following day with 0.1% Triton X-100. Subsequently, they were incubated for 2–3 h at room temperature (22 ± 1 °C) with respective primary antibodies prepared in 1% goat serum, followed by Alexa 488-conjugated goat anti-rabbit or goat anti-mouse IgG, Alexa 633-conjugated goat anti-rabbit or goat anti-mouse IgG, or both Alexa 488- and Alexa 633-conjugated IgG (Invitrogen, Thermo Fisher), for 1–2 h at room temperature. Finally, the cells were treated with Prolong Gold with DAPI (Invitrogen, Thermo Fisher). A Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) was used to analyze the cells.

Cells were seeded in 2-well Permanox Lab-Tek chamber slides (Nunc, Thermo Fisher), and were infected at 80–90% confluency with BoHV-1, BoHV-1YmVP8, or BoHV-1ΔUL49 at various MOIs. Cells were fixed at different times post-infection with 4% paraformaldehyde (Sigma-Aldrich Canada Ltd.) and washed with phosphate-buffered saline (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4 (PBS)). After washing, the cells were blocked overnight with 1% goat serum. The following day, the cells were treated with 0.1% Triton X-100 to permeabilize them. Subsequently, they were incubated with the corresponding primary antibodies prepared in 1% goat serum for 2–3 h, followed by Alexa 488-conjugated goat anti-rabbit or goat anti-mouse IgG, Alexa 633-conjugated goat anti-rabbit or goat anti-mouse IgG, or both Alexa 488- and Alexa 633-conjugated IgG (Invitrogen, Thermo Fisher), for 1–2 h at room temperature. Prolong Gold with DAPI (Invitrogen, Thermo Fisher) was used as a mounting medium and for staining of the nuclei of the cells after the antibody incubations. These cells were analysed with a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany), and Leica Application Suite X software was used to enlarge cells to indicate localisations in detail. Perinuclear localization of VP8 and/or VP22 was enumerated in 10 fields with 30–100 cells each.

Dissected guts from E16.5 embryos were digested with an enzyme mix of 1 mg/mL Dispase/Collagenase (Roche, 296638), at 37 °C. To obtain a cell suspension, culture medium containing 500 μL OptiMEM (Life technologies, 11058-021), 10 % foetal calf serum (FCS, PAA Labs, A15-649), 1 % L-glutamine (Life technologies, 25030-024), 1 % penicillin/streptomycin (Invitrogen, 15140-122), and 0.1 % ciprofloxacin (MP Biomedicals LLC, 199020) antibiotics was added to the digested tissue to aid dissociation with manual pipetting. The suspension was centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded and the cell pellet was re-suspended in fresh culture medium (for short-term culture) or simple OptiMEM (for FACS).
For FACS, samples were purified in a FACS ARIA II cell sorter (BD, specialised personnel in Francis Crick Institute, Mill Hill) to isolate YFP⁺ cells. Isolated YFP⁺ cells were collected in OptiMEM and processed for RNA extraction and RT-PCR. For short-term culture, cell suspensions were plated onto Fibronectin (Sigma, F1141)-coated, 8-well Lab-Tek permanox chamber (Thermo Fisher Scientific, 177445) slides. Cultures were then allowed to settle for approximately 15 hours in a 5 % CO₂ incubator before being fixed and processed for immunofluorescence.