Prolong diamond antifade mountant with dapi reagent

The uninfected and infected cells were grown on glass coverslips. The cells were washed with PBS, fixed with 4% paraformaldehyde for 10 minutes at room temperature, and then permeabilized with 0.5% Triton X-100 for 5 minutes and stored in PBS containing 0.2% Tween 20 (PBST) at 4°C. Prior to the immunofluorescent staining, the samples were incubated in blocking solution (10% donkey serum, 0.2% Tween 20, 0.2% Fish Skin gelatin in PBS) for 30 minutes. The antibodies were diluted in PBST. The primary antibodies that were used are anti-LANA (1/3000 dilution), anti-FLAG (1/100 for de novo infected cells and 1/1000 dilution for latently infected cells), anti-EZH2, anti-SUZ12, anti-BMI, anti-RING1B, anti-CYCT1, and anti-SPT5 (1/100 dilution). Secondary antibodies (goat anti-rat 633, goat anti-rabbit 568, and donkey anti-mouse 647) were used in 1/500 dilution. Primary antibody staining was performed for 2 hours followed by three times 5 minutes wash with PBST. This was followed by 45 minutes of incubation with secondary antibodies then three times 5 minutes wash with PBST. Thereafter, ProLong Diamond Antifade Mountant with DAPI reagent (Thermo Fisher Scientific) was applied and the coverslips were layered onto microscope slides. The samples were analyzed by confocal microscopy using 60x objective and the images were processed, analyzed, merged, and quantified with ImageJ software.

Female ticks fed on A. phagocytophilum-infected and uninfected sheep and fixed with 4% paraformaldehyde in 0.2 M sodium cacodylate buffer were embedded in paraffin and used to prepare sections on glass slides as previously described (Ayllón et al., 2015 (link)). The paraffin was removed from the sections through two washes in xylene and the sections were hydrated by successive 5 min washes with a graded series of 100, 96, and 65% ethanol and finally with distilled water. Next, the slides were treated with Proteinase K (Dako, Barcelona, Spain) for 7 min, washed with 0,1% PBS-Tween 20 (Sigma-Aldrich, St. Louis, MI, USA) and blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich) in PBS-Tween 20 during 1 h at room temperature. The slides were then incubated overnight at 4°C with mouse anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibodies (ab50567; Abcam, Cambridge, UK) diluted 1:100 in 2% BSA/PBS-Tween 20. Preimmune serum was used as control. After 3 washes with PBS-Tween 20, the slides were incubated for 1 h with rabbit anti-mouse IgG conjugated with FITC (Sigma-Aldrich) diluted 1:160 in 2% BSA/PBS-Tween 20. Finally, after two washes with PBS the slides were mounted on ProLong Diamond Antifade Mountant with DAPI reagent (Thermo Scientific, Madrid, Spain). The sections were examined using a Zeiss LSM 800 with Airyscan (Carl Zeiss, Oberkochen, Germany).