510 lscm

Mononuclear cells (MNCs) from the bone marrow of C57BL/6J mice were isolated via Ficoll density gradient centrifugation (Histopaque-1083, Sigma, Santa Clara, CA, USA) before being inoculated into culture flasks at a density of (3~5)×10⁶/ml and cultured with EGM-2 in 5% FBS (SingleQuots^®, Lonza, Basel, Switzerland) under an atmosphere of 95% humidity and 5% CO₂ at 37°C [15 (link)]. On day 4 of the culture, the culture fluid was replaced with fresh culture medium to remove the unattached cells. Cell harvesting was performed on day 7 of the culture. Double-positive staining of Ulex europaeus agglutinin-1(UEA-1) lectin and acetylated low-density lipoprotein (acLDL) was used for EPCs identification [15 (link)]. Briefly, the harvested cells were incubated with 7.5 μg/ml 1,1′-dioctadecyl-3,3,3′,3-tetramethylindocarbocyanine perchlorate(Dil)-labeled acLDL (Dil-acLDL, Molecular Probes, Eugene, OR, USA) at 37°C for 4 h and fixed with 4% paraformaldehyde for 10 min. After being washed, the cells were treated with 10.0 μg/ml fluorescein isothiocyanate (FITC)-labeled UEA-1 (FITC-UEA-1, Sigma, Santa Clara, CA, USA) for 30 min. Finally, the cells were treated with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 5 min before identification was performed using a laser scanning confocal microscope (LSCM 510, Carl Zeiss, Jena, Germany).

After fixed by embedding medium, trimmed terminal ileum of rats were fixed in liquid nitrogen and series of slices were prepared by a freezing microtome (Leica, Germany) at -20℃ with the thickness of 5 µm. Slides were then stored in sealed box at -80℃ for LSCM.
The prepared frozen slides were incubated at room temperature after the addition of 3% H2O2, blocked with 10% normal goat serum for 20 min, and incubated with mouse monoclonal anti- ZO-1 antibody (Invitrogen) overnight at 4℃. The sections were incubated at 37℃ for another 2 h after the addition of rabbit anti-mouse IgG antibody conjugated with fluorescein isothiocyanate (FITC) (Invitrogen, CA, USA). The slides were then sealed with 10% phosphate buffered saline (PBS) containing 90% glycerin and observed by a Laser scanning confocal microscope (LSCM-510, Carl Zeiss, Germany) with an excitation wavelength of 552 nm and an emission wavelength of 570 nm.