0.1 m sodium cacodylate

Infected cells were cytocentrifuged for staining with Gimenez and Diff-Quick (Dade Behring, Marburg, Germany) and were then examined under light microscope Leica^® DM LB2.
For the electron microscopy, 200 µL of 11 old days PL13-S2 co-culture were centrifuged for 15 min at 3000 rpm, then the supernatant was removed, and the pellet was fixed using 2.5% glutaraldehyde (Sigma Aldrich, Saint-Quentin-Fallavier, France) in 0.1 M sodium cacodylate buffer (Sigma Aldrich, Saint-Quentin-Fallavier, France) for 1 h. After fixation, the pellet was rinsed three times with 0.1 M sodium cacodylate (5 min each) to remove residual fixative. The graded ethanol concentrations (25% for 5 min; 50% for 5 min; 70% for 5 min; 85% for 5 min; 95% for 5 min (twice); 100% ethanol for 10 min (three times) was used for sample dehydration. Finally, the pellet was incubated for 5 min in an ethanol/Hexamethyldisilazane (Sigma Aldrich, Saint-Quentin-Fallavier, France) (1:2) mixture, then in pure HMDS. The mixture was cytocentrifuged for 5 min at 2000 rpm and the glass slide allowed to air dry for 30 min before observation. The examination was performed using a TM4000 Plus^TM (Hitachi, Tokyo, Japan) scanning electron microscope operated at 10 kV in BSE mode at magnifications ranging from X200 to X3000.

Cells were fixed in Karnovsky fixative (2% paraformaldehyde and 2.5% Glutaraldehyde in 0.1 M sodium cacodylate buffer (Sigma-Aldrich) for 20 mins, and post-fixed in 1% osmium tetroxide (Sigma-Aldrich) dissolved in 0.1 M sodium cacodylate (Sigma-Aldrich) buffer pH 7.4. Fixed cells were dehydrated in increasing concentrations (70–100%) of ethanol and acetone, and then embedded in BEEM resin capsules and polymerized overnight at 70°C. Ultrathin sections were cut at 80-100 nm and mounted on 300 mesh copper grids, stained with lead citrate (Electron Microscopy Sciences, Hatfield, PA), and observed using an FEI Tecnai G2 Spirit transmission electron microscope (FEI, Hillsboro, OR) operating at 80 KV.