Alexafluor 555 labeled donkey polyclonal anti rabbit igg

Frozen aortic root sections were equilibrated to room temperature, rehydrated with phosphate buffered saline (PBS), permeabilized with 10% DMSO, and blocked with 0.05% casein in PBS. Sections were then stained overnight at 4 °C in a humidified chamber with rat monoclonal anti-mouse CD68 (1:400, BioLegend, clone FA-11) and rabbit polyclonal anti-mouse IL-10 (1:25, Bioss, bs-0698R) or rabbit polyclonal anti-mouse iNOS (1:50, Proteintech, 18985-1-AP). Sections were washed twice with PBS supplemented with 0.05% Tween (PBS-T) and then stained for 1 h at room temperature in a humidified chamber with AlexaFluor-488-labeled donkey polyclonal anti-rat IgG (1:500, Invitrogen, A21208) and AlexaFluor-555-labeled donkey polyclonal anti-rabbit IgG (1:500, Invitrogen, A31572). Sections were washed twice with PBS-T and once with PBS before mounting with ProLong Gold Antifade Mountant with DAPI (Invitrogen). Images were acquired using a DMI8 inverted fluorescence microscope (Leica Microsystems) and analyzed using QuPath (v0.5.0).

Human cryosections from the aorta of a 76-year-old male with atherosclerosis were purchased from OriGene (CAT#: CS611744). Sections were stained as above with minor alterations. After permeabilization and blocking, sections were incubated with WT IL-10 or 2D03-IL-10 (50 μg ml⁻¹ IL-10 basis) at room temperature in a humidified chamber for 2 hr. Washes were performed with PBS supplemented with 0.025% Tween for 1 min each to minimize disruption of plaque lipids. Sections were then stained overnight at 4 °C in a humidified chamber with mouse monoclonal anti-human CD68 (hCD68, 1:400, BioLegend, clone Y1/82A) and rabbit polyclonal anti-mouse IL-10 (mIL-10, 1:25, Bioss, bs-0698R). The following day, sections were stained for 1 h at room temperature in a humidified chamber with AlexaFluor-488-labeled donkey polyclonal anti-mouse IgG (1:500, Invitrogen, A21202) and AlexaFluor-555-labeled donkey polyclonal anti-rabbit IgG (1:500, Invitrogen, A31572). Sections were mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen). Images were acquired using a DMI8 inverted fluorescence microscope (Leica Microsystems) and formatted using QuPath (v0.5.0).