Immunofluorescence microscope

Early and late EPC colonies were prepared for the lectin binding and uptake studies using the following methods. First, EPCs were incubated with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyindocarbocyanide-labeled acetylated low-density lipoprotein (Dil-Ac-LDL; Biomedical Technologies Inc., Stoughton, USA) at 10 μg/mL in EGM-2 medium at 37°C for 4 h. They were then washed three times with PBS and fixed with 4% formaldehyde in PBS for 20 min at room temperature. Subsequently, the EPCs were incubated with 200 μL of mouse anti-human UEA-1 (Ulex lectin) antibody-conjugated with fluorescein isothiocyanate (FITC) (Sigma, St. Louis, USA) at 4°C for 1 h and then washed three times with PBS.
Otherwise, early and late EPCs were washed twice with PBS and fixed with 2% paraformaldehyde in PBS and then incubated for 18 h at 4°C in the dark with the following antibodies: a phycoerythrin- (PE-) conjugated anti-human CD34 antibody (BD Bioscience, USA), a FITC-conjugated anti-human CD45 antibody, and a PE-conjugated anti-kinase domain receptor (KDR) antibody (R&D Systems, USA). Stained colony dishes were observed with an immunofluorescence microscope (Keyence, Osaka, Japan).

Localisation of claudins-1 and -4, occludin and ZO-1 on Caco-2 cells was assessed by immunofluorescence microscopy. Caco-2 cells were seeded on Transwell filters (diameter, 12 mm; pore size, 0.4 µm; Corning). After treatment with thiostrepton (100 µM) or no treatment for 24 h, the Caco-2 cells were fixed with methanol–acetone (1:1 v/v) for 5 min and then permeabilised with 0.2% Triton X-100 in PBS for 10 min. After non-specific binding had been blocked by incubation with 1% BSA in TBS buffer (20 mM Tris-HCl, pH 7.4, 40 mM NaCl) containing 0.05% Tween-20 (T-TBS) for 1 h, the cells were incubated with anti-claudin-1, -claudin-4, -occludin and -ZO-1 antibodies in 1% BSA in T-TBS for 1 h. After incubation of the cells with fluorescent secondary antibodies for 1 h, the immunofluorescence images were observed under an immunofluorescence microscope (Keyence, Tokyo, Japan). For visualisation of biotin passing through the TJ, Caco-2 cells treated with 100 µM thiostrepton or left untreated were apically labelled with sulfo-NHS-SS-biotin (606.7 Da) for 10 min. The cells were then fixed, and anti-ZO-1 antibody and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) were employed for immunofluorescence staining of the TJ or nucleus, respectively.