Dmil led phase contrast microscope

When applying a cell-seeded stent in vivo, the blood vessel inner lumen outside the mesh framework will be denuded. These areas of native tissue without ECs need to be covered by endothelial cells growing out from the stent. To simulate this situation in vitro, cell-seeded stents were placed in a gelatin-coated culture flask to study cell outgrowth. Cell coverage of the gelatin-coated surfaces was qualitatively assessed once a day using a Leica DMIL LED phase contrast microscope for 7 days (Fig. 5).Fig. 5

ECs seeded onto gelatin-coated stents (I) and (III) are still proliferative and able to endothelialize a surface beyond the stent surface. A, B phase contrast images after 48 h in culture

8505C cells were treated with 5 ng/mL TGF-β1 for 48 h. Morphological changes in the 8505C cells were observed under a Leica DMIL LED phase contrast microscope with an attached EC3 camera (Leica, Germany). The photographs were taken at 200 x magnification. Immunofluorescence staining was used to detect E-cadherin, cytokeratin, vimentin, and F-actin in the 8505C cells as the manufacture’s instruction. The fluorescent images were obtained by confocal laser scanning microscope (Zeiss LSM 510, Oberkochen, Germany).
After 8505 cells were maintained in media supplemented with 5 ng/mL TGF-β1 with or without 60 μM EGCG for 48 h. 8505C cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde. After
permeabilization with 0.5% Triton X-100 and blocking in 5% bovine serum albumin (BSA), the cells were incubated
with primary E-cadherin or vimentin antibodies overnight at 4°C then incubated with fluorescence-conjugated secondary antibodies at room temperature for 1 h. The nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) for 5 min. Images were pseudocolored using a Zeiss Instruments confocal microscope.