Lsm 780 confocal scanning microscope

This study cultured bEND.3 cells (4.5 × 10⁴ cells) on cover glass for 7 days until confluent. After TNF-α treatment, the cells were washed in warm PBS and fixed in 1.75% paraformaldehyde for 15 min. The cells were subsequently incubated with PBS containing 0.1% Triton X-100 for 10 min (except for those subjected to immunofluorescence analysis for FN) and then blocked with 1% bovine serum albumin in PBS for 30 min at room temperature. After blocking, the cells were incubated with primary antibodies against FN (#AB2033, 1:200, Merck Millipore), ZO-1 (#61–7300 or #33–9100, 1:200, Thermo Fisher Invitrogen, Waltham, MA, USA), activated β1 integrin (clone HUTS-4, 1:100, Merck Millipore), β3 integrin (#13166, 1:200, Cell signaling, Denver, MA, USA), FAK (#3285, 1:200, Cell Signaling), and vascular endothelial (VE)-cadherin (#36–1900, 1:200, Thermo Fisher Invitrogen) overnight at 4 °C followed by Alexa Fluor 488– and 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 h at room temperature. Some cells were coincubated with Alexa Fluor 488–conjugated phalloidin (1:200, Thermo Fisher Invitrogen). Confocal images were captured using a Zeiss LSM 780 confocal scanning microscope (Oberkochen, Germany), and the images were processed using Photoshop CS6 (Adobe, San Jose, CA, USA).

To locate MFG-E8 and phosphorylated NF-κB p65 in the mouse and human arteries, the paraffinized sections were deparaffinized, permeabilized with 0.1% Triton-X 100 in phosphate-buffered saline (PBS-T) for 10 min, blocked with 5% normal goat serum in PBS-T, and incubated with primary antibodies against MFG-E8 (1:200, R&D Systems) and NF-κB p65 phosphorylated at Ser536 (1:200, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C followed by an additional incubation of Alexa Fluor 488– and 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen, Waltham, MA, USA) for 1 h at room temperature. VSMCs derived from young and aged aortas were washed in warm PBS and fixed in 1.75% paraformaldehyde for 15 min after the various treatments. After blocking, the cells were incubated with primary antibodies against MFG-E8 and NF-κB p65 phosphorylated at Ser536 followed by the appropriate secondary antibodies. Confocal images were captured using a Zeiss LSM 780 confocal scanning microscope (Oberkochen, Germany), and the images were processed in Photoshop CS6 (Adobe, San Jose, CA, USA).