Atto425 conjugated streptavidin

NIH cells (Fig. 2) were seeded onto cover slips placed in 24-well plates. After 24 h, the medium was changed to new medium containing 360 μM oleic acid. After 20 h, the cells were fixed with 2% formaldehyde for 10 min and stored at 4°C in PBS. For immunolabeling, the cells were treated with Avidin/Biotin Blocking kit (Vector Laboratories), and 1% BSA in PBS for 20 min. The cells were then incubated 2h at room temperature with mouse anti-vinculin (1:100, clone SPM227, Abcam), followed by 30 min biotinylated monovalent donkey anti-mouse IgG (1:200, Jackson ImmunoResearch) and 30 min with Atto425-conjugated streptavidin (1:200, ATTO-TEC). The cells were then incubated for 2h at room temperature with guinea pig anti-ADRP (1:400, Fitzgerald), rabbit anti-mouse syntaxin 5 (1:150, Synaptic System) and rat anti-mouse LAMP1 (1:100, clone 1D4B, Abcam) followed by 30 min incubation with goat anti-guinea pig AF488 (1:400, Life Technologies), goat anti-rabbit Cy3 (1:250, Jackson ImmunoResearch), mouse anti-rat PerCP-eFluor710 (1:100, Affymetrix) and phalloidin-Atto594 (5 μL/mL, Sigma-Aldrich); in the last 5 min, 3 drops of DAPI (2 μg/mL) was added to each well. All incubation steps contained 0.1% saponin and all washing steps contained 0.05% saponin for permeabilization. The cover slips were mounted onto microscope slides with Prolong Gold antifade reagent (Life Technologies).

Arterial tissue sections (S3 Fig.) were incubated overnight at 4°C with rat anti-mouse CD18 (1:50, clone C71/16, Cedarlane), Cy3-conjugated mouse-anti human α-actin (1:15000, clone 1A4, Sigma-Aldrich), goat anti-mouse/human ApoB (1:100, R&D systems), and rabbit anti-Ki67 (1:100, clone SP6, GeneTex). The sections were washed and incubated with F(ab)2 AF594-conjugated monovalent donkey anti-goat (1:200, Jackson ImmunoResearch,) and biotinylated donkey anti-rabbit (1:200, Jackson ImmunoResearch) at room temperature for 45 min, followed by Atto425-conjugated streptavidin (1:200, ATTO-TEC) and AF488-conjugated goat anti-mouse CD31 (1:150, R&D systems) at room temperature for 45 min. Nuclei were stained with DAPI (2 μg/ml for 4 min) and the slides were mounted with ProlongGold mounting media (Life Technologies).

Spleen tissue sections with germinal centers (Fig. 3 and S4 Fig.) were incubated for 2h at 20°C with rat anti-mouse Foxp3 (1:100, Clone FJK-16s, Affymetrix) and hamster anti-MFGE8 (1:100, clone 18A2-G10, MBL) followed by 45 min with F(ab)2 AF594-conjugated donkey anti-rat IgG (1:400, Jackson ImmunoResearch) and Cy3-conjugated goat anti-Armenian hamster IgG (1:400, Jackson ImmunoResearch). The sections were then blocked 15 min with nonspecific rat IgG2a (1:30, Clone R35-95, BD Biosciences) and incubated 60 min with AF488-conjugated rat anti-human/mouse B220 (1:100, clone RA3-6B2, Affymetrix), biotinylated rat anti-mouse CD4 (1:100, clone RM4-5, Affymetrix) and PerCP-eFluor710-conjugated rat anti-mouse IgD (1:100, clone 11–26c, Biolegend). The sections were then incubated 30 min with Atto425-conjugated streptavidin (1:300, ATTO-TEC). Nuclei were stained with DAPI (2 μg/ml for 4 min) and the slides were mounted with ProlongGold mounting media (Life Technologies).