*4.4. Transmission Electronic Microscopy*

Trophoblastic cells were seeded for 15 h post isolation and incubated with LPV or control DMSO for 6, 24 or 48 h. Cells were collected after each time by trypsin-EDTA, centrifuged 5 min at 1400× *g*, and washed twice in PB (0.05 M PIPES [piperazine-N,N' bis(2-ethanesulfonic acid)] buffer, 5 mM CaCl2, pH 7.3) for 10 min, then centrifuged 5 min at 1400× *g*. Each sample was fixed during 45 min at room temperature protected from light in PB containing 2.5% glutaraldehyde and 2% paraformaldehyde. After 5 min of centrifugation at 1400× *g*, samples were washed twice for 10 min in PB and then post-fixed first in PB–1% osmium tetroxide (45 min at 4 ◦C) and then in 1% aqueous uranyl acetate solution for 2 h at room temperature. Samples were then dehydrated in increasing concentrations of ethanol (30%, 50%, 70%, 95% and 100%) followed by ethanol/propylene oxide (1/1 (vol/vol)) and propylene oxide and were finally embedded in Epon epoxy resin. Ultrathin sections (80 nm of thickness) were performed with a Leica ultracut S microtome fitted with a diamond knife (Diatome histoknife Jumbo or Diathome ultrathin). Theses sections were stained with lead citrate and placed on copper grids. The sections were analyzed at 80 kV with a Jeol electron transmission microscope (JEM-100S transmission electron microscope, Croisy sur Seine, France). Acquisitions were made with Gatan software (Gatan Microscopy Suite ®, Gatan Inc., AMETEK, Pleasanton, CA, USA).
