Tcs sp5 2 scanning confocal microscope

HUVECs were cultured in plastic dishes that were obtained from ibidi, 35 mm round, appropriate for microscopy, previously coated with collagen type I. For internalization experiments, cells were washed 3 times with blocking buffer, treated for 30 min with vehicle or inhibitors in blocking buffer, transferred to 37 °C and incubated with 50 μg/mL fluorescein isothiocyanate-conjugated transferrin (Invitrogen). Then, the samples were washed 3 times with Ca²⁺/Mg²⁺ HBSS, fixed in 3.7% PFA for 20 min and analyzed by microscopy. Images of the cells were captured using a Leica TCS SP5 II scanning confocal microscope and a Leica 63X HCX PL APO 1.4 NA objective. Imaging data were subsequently processed using the LAS AF software, according to the manufacturer’s guidelines.

HUVECs were cultured in 35-mm diameter plastic dishes (appropriate for microscopy, by Ibidi), coated with collagen type I. To monitor the internalisation fate of cell surface pool of VEGFR2, 2 h serum-starved HUVECs were transferred to 4 °C for 30 min and incubated for 1 h with 10 μg/ml of mouse anti-VEGFR2 extracellular domain antibodies. Cells were treated for 30 min with vehicle (DMSO) or dynasore (100 μM) and transferred to 37 °C for 15 min in the presence of 50 μg/ml fluorescein isothiocyanate-conjugated transferrin and VEGF (50 ng/ml). Cells were acid-washed twice (ice cold M199 medium, pH 2.0), fixed and processed for immunofluorescence microscopy. The above protocol was also applied to siRNAs treated cells.
Indirect immunofluorescence and analysis by confocal microscopy was employed as previously described48 (link). Images were captured using a Leica TCS SP5 II scanning confocal microscope and a Leica 63X HCX PL APO 1.4 NA objective. Data were subsequently processed in LAS AF according to the manufacturer guidelines.

Cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature followed by permeabilization with 0.2% Triton X-100 in PBS for 5 min. For DNase I digestion, after fixation, and permeabilization, cells were treated with 500 units/mL RNase-Free DNase I (Qiagen, Cat. No. 79254) for 1 h at 37 °C. After blocking with 1% BSA in PBS, cells were incubated with primary antibody overnight at 4 °C and Alexa-Fluor-conjugated secondary antibody (Life Technologies). Fluorescent images were captured using a Leica TCS SP5 II scanning confocal microscope.