Tcs sp5 dmi6000 confocal microscope

Confocal images were recorded using an Olympus Fluoview FV1000 confocal microscope (Olympus Life Science Europa, Hamburg, Germany), a Leica TCS SP5 DMI6000 confocal microscope (Leica Microsystems, Wetzlar, Germany), a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) and a Zeiss LSM 780 (Carl Zeiss Jena GmbH, Jena, Germany).

HUVECs were seeded on gelatin-coated glass coverslips in a 12-well plate or IBIDI slides (ibidi, Martinsried, Germany) and cultured to confluency. Upon treatment, cells were washed with DPBS, fixed in PFA for 5 min at room temperature, blocked and permeabilized (0.5% BSA, 5% donkey serum, 0.1% TritonX-100 in DPBS) for 30 min. Cells were incubated with primary antibodies overnight at 4 °C, washed with DPBS 4–5 times and incubated with relevant fluorescent-conjugated secondary antibodies. DAPI (4,6-diamidino-2-phenylindole) was used to counterstain nuclei. The stained cells were mounted with ‘Prolong’ antifade mounting medium (Molecular Probes (Basel, Switzerland) and imaged using a Leica TCS SP5 DMI6000 confocal microscope (Leica Microsystems, Wetzlar, Germany).

The localization of Visudyne® within the artery was assessed on histological sections (5 μm) using a Leica TCS SP5 DMI6000 confocal microscope (Leica Microsystems) equipped with Leica plan apo 20 × (numerical aperture 0.7) dry objective. The sections were illuminated with 405 nm Diode laser. Images were collected in the fluorescence emission range of 650–750 nm. Elastin auto fluorescence was co-detected upon excitation with 488-nm laser light. Pictures were taken by using Leica Application Suite Advanced Fluorescence (LASAF) software (Leica Microsystems). Mean fluorescence intensity was quantified and normalized with respect to the area of tissue using the ImageJ software as previously described (Jensen, 2013 (link)).