Opal polymer horseradish peroxidase

Consecutive staining was performed by heat-induced antigen retrieval followed by incubation with primary antibody (anti-CD163, 1:200, ab182422, Abcam, Cambridge, MA, USA). The signal was amplified and detected with Opal™ polymer horseradish peroxidase and Opal 570 (1:100, Akoya Biosciences, Japan). The sections were then subjected to heat-induced antibody stripping and incubated with the next antibody (anti-DC-SIGN was used at a dilution of 1:100 and detected with Opal 520; then, anti-CD68 was used at a dilution of 1:200 and detected with Opal 620; and finally, anti-CD14 was used at a dilution of 1:200 and detected with Opal 650) and spectral DAPI (anti-DC-SIGN, ab218419; anti-CD68, ab213363; anti-CD14, ab133503, Abcam, Cambridge, MA, USA). All Opal reagents were used at a dilution of 1:100 (Opal 520, Opal 620 and Opal 650, Akoya Biosciences, Japan). The slides were finally mounted in antifade mounting medium (Akoya Biosciences, Japan) and scanned with a Vectra Polaris (Akoya Biosciences, Japan). The biomarkers to define macrophage populations are summarized in Table 3. Meanwhile, the biomarkers of M1 macrophage were recorded as the control. Digital pathology methods were used to determine the density of positive cells. All analyses were performed with Image-pro Plus.