Prolong gold antifade mounting medium

Cells were seeded onto 11 mm coverslips placed in wells of a 24-well plate to 65–75% confluency. Inhibitors were added as described, and the cells were labelled with 2 µM pacSph in serum-free DMEM for the indicated times. Subsequently, cells were washed with 1 mL PBS three times at RT. Cells were overlaid with 0.5 mL of cold imaging buffer (20 mM HEPES, 115 mM NaCL, 1.2 mM MgCl₂, 1.2 mM glucose and 1.8 mM CaCl₂, pH 7.4/NaOH), and UV-irradiated (λ~365 nm) on ice for 5 min. Cells were immediately fixed with pre-cooled MeOH at −20 °C for 20 min. Non-cross-linked lipids were extracted by washing three times with 0.78 mL of CHCl₃/MeOH/AcOH (10:55:0.75) (v/v) and twice with PBS. Cells were then incubated with 50 μL of click mixture (1 µL 2 mM Alexa-555-azide, 125 µL 10 mM Cu(I)BF₄ in acetonitril and 0.5 mL PBS) for 1 h at room temperature in the dark. Cells were then washed with PBS and incubated with 50 μL of primary α-LAMP1 antibody (1:200 in PBS supplemented with 2% BSA and 0.3% Triton X-100) for 1 h in the dark. Coverslips were briefly washed with PBS and incubated with secondary antibody (α-rabbit conjugated to Alexa Fluor 488, 1:800) for 30 min, washed briefly with PBS, and mounted in ProLong Gold Antifade mounting medium (Cell Signalling, Danvers, MA, USA). Microscopy images were captured using a confocal laser-scanning microscope (Zeiss LSM800) with a 63 × oil objective.

Tissue sections were prepared by embedding in OCT (Thermo Fisher Scientific, Waltham, MA) followed by freezing at − 80°C. Vessel leakiness was evaluated following tail‐vein injection of 100 μL of Dextran Texas Red (Invitrogen). Vessel integrity was assessed after tail‐vein injection of 50 μL of lectin labelled with FITC (Vector Labs, Burlingame, CA). Apoptotic cells were visualized using an anti‐rabbit cleaved caspase 3 primary antibody (Cell Signaling Technology, Danvers, MA) and an goat‐anti‐rabbit Alexa Fluor 594 secondary antibody (Cell Signaling Technology); proliferating cells were visualized using an anti‐mouse Ki67 primary antibody (Cell Signaling Technology) and an goat‐anti‐mouse Alexa Fluor 488 secondary antibody (Cell Signaling Technology); DAPI containing ProLong Gold Antifade Mounting Medium (Cell Signaling Technology) was used to visualize the nuclei and mount the slide.

Cells were grown onto 11 mm coverslips in a 24-well plate to be 65–75% confluent. They were fixed in 4% formaldehyde for 20 min at RT and were subsequently rinsed twice with PBS. Formaldehyde was quenched with 20 mM glycine in PBS for 10 min at RT and washed again with PBS. The first antibody was added in 1% BSA/0.3% Triton/PBS and was incubated for 1 h at RT. Coverslips were washed briefly with PBS, and a secondary antibody was added (1:800, Alexa Fluor 488 or Alexa Fluor 594 or Alexa Fluor 647, Cell Signalling) in the same antibody dilution solution and was incubated for 30 min at RT. Coverslips were washed with PBS and were mounted in ProLong Gold Antifade mounting medium (Cell Signalling). Microscopy images were captured using confocal microscopy (Zeiss LSM800) with a 63 × oil objective.