Goat anti ve cadherin

sEND.1 cells were cultured in confocal dishes (180504, Cellvis) overnight and treated with or without sodium lactate (10 mM, L7022, Sigma), IFNγ (100 ng/mL, 315-05, Peprotech), Lactate dehydrogenase A (LDHA) inhibitor (GNE140, 5 μM, HY-100742, MCE), and bafilomycin (Baf, 100 nM, 1334, TOCRIS) for 12 h. Cells were then fixed with 4% paraformaldehyde for 15 min and permeabilised with 0.5% Triton X-100 for 5 min. Next, cells were blocked with 3% (w/v) BSA in phosphate buffered saline (PBS) for 30 min at 37 °C and then incubated with Goat-anti-VE-cadherin (0.2 μg/mL, R&D Systems Cat# AF1002, RRID: AB_2077789) and Rabbit-anti-LAMP (1:100, Cell Signalling Technology Cat# 9091, RRID: AB_2687579) for 1 h. Next, these were labelled with Alexa-Fluor-555 donkey-anti-goat second antibody (1:400, Thermo Fisher Scientific Cat# A-21432, RRID: AB_2535853) and Alexa-Fluor-488 donkey anti-rabbit second antibody (1:400, Molecular Probes Cat# A-21206, RRID: AB_2535792) for 50 min at 37 °C, and stained with 4',6-diamidino-2-phenylindole (17985-50, Electron Microscopy Sciences) for 10 min. The images were captured using a spinning-disk confocal microscope.

Cells were seeded into wells of a 24-well plate containing glass slides and cultured until confluence. Cells were fixed with 4% PFA for 10 min at room temperature, blocked in blocking solution (PBS, 0.05% Tween 20, 0.1% Triton-X, 5% donkey serum, 5% BSA) and stained with primary antibodies (in blocking solution) overnight at 4 °C, followed by washing in PBS and incubation with secondary antibodies for 1 h at room temperature. After washing, glass slides were mounted using Mowiol (Sigma-Aldrich). Primary antibodies were: rabbit anti-MEF2C (1:400, Cell Signaling, 5030), mouse anti-BCL6 (1:100, Santa Cruz, sc-7388) and goat anti-VE-cadherin (1:100, R&D, AF938). Secondary antibodies were: donkey anti-goat AlexaFluor594, donkey anti-mouse AlexaFluor488, donkey anti-rabbit AlexaFluor647 (1:1000, all from Life Technologies). Confocal images were taken with an LSM780 microscope (Zeiss).

The visualization of cells (HCAECs, monocytes/macrophages, HCASMCs), and assessment of LDL uptake was carried out using non-immunofluorescence and immunofluorescence confocal microscopy. For the non-immunofluorescence microscopy, cells were pre-stained with calcein AM (2 μM) for 10 minutes. The culture models were then plated in 35-mm glass-bottom microwell dishes (Cat#: P35G-1.5-14-C, MatTek, Ashland, MA; 100μL/well). For the immunofluorescence microscopy, the plated co-culture models were cultured for 24 hours, fixed with cold methanol (-20°C for 20 min), washed with PBS (phosphate buffered saline), and blocked with 5% donkey serum dissolved in PBS. Subsequently, the cultures were incubated at 4°C overnight with three primary antibodies: (i) Goat anti-VE-cadherin (R&D Systems, Cat#: AF938, 1:500) for ECs, (ii) Rabbit anti-CD68 (Abcam, Cat#: ab125047, 1:500) for macrophages and (iii) Mouse anti-α-smooth muscle actin (R&D Systems, Cat#: MAB1420,1:500). After 24 hours, dye-conjugated secondary antibodies (Donkey anti-mouse/rabbit/goat-Alexa 488- or 594, Invitrogen, Cat#: A32766, A32814, A32790, A32744, or A32754, 1:2000) were added for 1 hour and nuclei were stained with 4′,6 diamidino-2-phenylindole (DAPI) for 10 minutes.