HUVECs were grown to confluence on 0.1% gelatin-coated coverslips. EVs isolated from plasma were stained with 5 μM carboxyfluorescein succinimidyl ester (#423801, BioLegend) for 30 minutes at RT. After washing two times with PBS, EVs (~1×107) were applied on HUVECs monolayer and incubated for 6 hours. EVs that were not taken up by HUVECs were removed by washing with PBS. Cells were then fixed with 4% PFA for 10 minutes at RT, washed, and permeabilized with PBS+0.05% Triton. After blocking with 10% donkey serum, the coverslips were incubated with rabbit anti-VE-cadherin antibody (#2500S, CST) overnight at 4°C, followed by incubation with donkey anti-rabbit AlexaFluro 568 (A10042, Invitrogen) for 1 hour at RT. ProLong diamond mounting medium with DAPI (#P36962, Life Technologies) was used for nuclei staining and mounting. Images were captured using Leica SP8 Spectral Inverted Laser Scanning Confocal Microscope. The discontinuity of VE-cadherin staining was analyzed using Imaris software (Bitplane).
Sp8 spectral inverted laser scanning confocal microscope
Manufactured by Leica
The SP8 Spectral Inverted Laser Scanning Confocal Microscope is a high-performance imaging system designed for advanced microscopy applications. It features a spectral detection system that enables the simultaneous acquisition of multiple fluorescence channels, allowing for detailed analysis of complex samples. The SP8 operates on an inverted configuration, providing a stable platform for live-cell imaging and sample manipulation.
Automatically generated - may contain errors
3 protocols using sp8 spectral inverted laser scanning confocal microscope
1
EV Uptake and VE-Cadherin Disruption in HUVECs
2
Immunohistochemical Analysis of DHHC21 and α1AR
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Human renal arteries were fixed in 10% formalin for 48 h, paraffin-embedded and sectioned. Slides were then deparaffinized, rehydrated and permeabilized with PBS containing 0.05% Triton X-10022 (link). After blocking, slides were labelled with rabbit anti-DHHC21 and goat anti-α1AR antibodies (1:100) overnight at 4 °C. After washing, secondary antibody incubation was done with donkey anti-rabbit Alexa Fluor 488 and donkey anti-goat Alexa Fluor 568 (1:500). After mounting with ProLong Diamond mounting medium with DAPI, slides were imaged with Leica SP8 spectral inverted laser scanning confocal microscope.
3
Neutrophil Adhesion to Endothelial Cells Modulated by EVs
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MLMVECs were pre-stimulated with 100 ng/ml TNF-α for 4 h and rinsed three times with warm HBSS. EVs from different groups were normalized to the same concentration. 1 × 105 neutrophils were incubated with 100 μl EVs (∼108) for 1 h at 37°C with or without the presence of complement inhibitor DAF (10 μg/ml). Neutrophils were then pelleted at 300 g for 5 min, resuspended, and applied on MLMVECs. After 1 h incubation, non-adherent neutrophils were washed off with HBSS for three times (Chatterjee, Yang, Ma, Cha, et al., 2020 (link)). Coverslips were fixed, permeabilized with PBS+0.1% Triton X, and blocked with 10% donkey serum. MLMVECs and neutrophils were labelled with goat anti-mouse ICAM-1 antibody and rat anti-mouse neutrophil antibody respectively overnight at 4°C. After washing three times, donkey-anti-goat AlexaFluor568 and donkey-anti rat AlexaFluor488 were added on the coverslips for 1 h. Prolong Diamond Mounting Medium was used to for nuclei staining and mounting. Images were captured using Leica SP8 Spectral Inverted Laser Scanning Confocal Microscope.
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