TEM Preparation of Human LEC Cultured on Hydrogels

Human LECs cultured on hydrogels for 6 and 12 hours were prepared for TEM samples as previously described.^{25 (link)} Briefly, cells were fixed with 3.0% formaldehyde, 1.5% glutaraldehyde in 0.1 M Na cacodylate, 5mM Ca²⁺ and 2.5% sucrose at room temperature for 1 hr and washed three times in 0.1 M cacodylate/2.5% sucrose pH 7.4 for 15 minutes each. The cells were post-fixed with Palade’s OsO₄ on ice for 1 hour, rinsed with Kellenberger’s uranyl acetate and then processed conventionally through Epon embedding on a 16-well Lab-Tek chamber slide (NUNC). Serial sections were cut, mounted onto copper grids, and viewed using a Phillips EM 410 transmission electron microscope (FEI, Hillsboro, OR). Images were captured with a FEI Eagle 2k camera.