Fv1200 spectral inverted laser scanning confocal microscope

HUVECs were grown to 100% confluence on 0.1% gelatin-coated coverslips, then treated with rhSDC ectodomain fragments or vehicle for 2, 4, 6, 8 or 25 minutes before being fixed 2% PFA for 10 minutes then 4% PFA for 10 minutes. HUVECs were washed with 3% BSA/PBS, then permeabilized with 0.1% triton-x in 3%BSA/PBS for 10 minutes. Following further washes, the cells were then blocked with 10% donkey serum in 3%BSA/PBS with 0.01% tween-20 for 60 minutes. HUVECs were then stained with anti-VE-cadherin (1:200, overnight 4°C). The next day, HUVECs were washed and then stained with AlexaFluor 647-conjugated phalloidin (1:250) and AF488-conjugated donkey anti-rabbit (1:500) for 60 minutes at room temperature. Further washes were followed by staining with DAPI hydrochloride (10 μg/ml) for 15 minutes. HUVECs were washed for the final time before the coverslips were mounted on slides with ProLong diamond antifade mountant. Images were taken with a x60 objective using an Olympus FV1200 spectral inverted laser scanning confocal microscope with associated Olympus FluoView software. A total of 6–8 x 1 μm slices were taken and used to create a maximum projection Z-stack.

For sectioning of slices on the vibratome, the left upper lobe was isolated, and the airways filled with 3% w/v agarose in Dulbecco’s Modified Eagle Media (DMEM) via the bronchus. The lobe was placed on ice and the agarose allowed to set for 30 minutes before small sections of the lobe were cut and prepared for vibratome sectioning. Sections (200 μm thick) were cut on a vibratome in Hanks balanced salt solution. Sections were then fixed for 24 hours in 10% neutral buffered formalin, before being blocked and permeabilized with 10% donkey serum, 0.1% triton X, 0.1% tween in PBS for 60 minutes. Sections were stained with DAPI hydrochloride (10 μg/ml) as well as primary antibodies for CD31/PECAM-1 (1:250) and either SDC-1 (1:200), SDC-2 (1:100,), SDC-3 (1:100), SDC-4 (1:100) for 2 hours at room temperature. Sections were washed 5 x 5 minutes in 0.1% tween in PBS, then incubated for 2 hours at room temperature with AF488-conjugated donkey anti-mouse (1:500) and AF647-conjugated goat anti-Armenian hamster (1:250) secondary antibodies. Following 5 x 5-minute washes in 0.1% tween in PBS, the sections were mounted on slides with ProLong diamond antifade mountant. Images were taken using an Olympus FV1200 spectral inverted laser scanning confocal microscope with associated Olympus FluoView software. Approximately 15 slices were taken and used to create a maximum projection Z-stack.

Human lung microvascular endothelial cells (HLMVECs) were treated with vehicle or thrombin (10 U/ml) for 1 hour and subsequently fixed. Cells were stained for S1ED (1:200), S2ED (1:100), S3ED (1:100) or S4ED (1:100) for 1 hour at room temperature then washed before secondary antibody (AF488; 1:500; 1 hour room temparature) was applied. Following washes, coverslips were mounted on slides with ProLong diamond antifade mountant. Images were taken using an Olympus FV1200 spectral inverted laser scanning confocal microscope with associated Olympus FluoView software. A total of 6–8 x 1 μm slices were taken and used to create a maximum projection Z-stack.