Brilliant violet 605 anti mouse cd206

Interstitial cells were collected and stained according to the manufacturer’s instructions. The following antibodies were used: anti-mouse CD16/32 (Biolegend, 156604), Zombie-APC/CY7 (Biolegend, 423106), FITC anti-mouse CD45 (Biolegend, 103108), PE anti-mouse F4-80 (Biolegend, Clone: BM8, 123110), PE/cyanine5 anti-mouse CD86 (Biolegend, 105016), and Brilliant Violet 605™ anti-mouse CD206 (Biolegend, 141721). The labelled cells were detected using a flow cytometer (Cytek Aurora or BD LSRII). Data were analysed using FlowJo software (Version 10.4.0, USA).

Explanted mice ears where cut by a biopsy pouch centered in the middle zone (diameter 8 mm), washed twice with PBS, embedded at the hedge in O.C.T. (Tissue-Tek O.C.T. Compound, Sakura Finetek), and instantly frozen in liquid nitrogen. 8-μm thick slides were obtained cutting ears block with a cryostat at −20 °C. For H&E staining, slides were thawed, hydrated, washed and stained with hematoxylin and eosin (Sigma-Aldrich). Immunofluorescence staining has been performed on consecutive ear sections. Briefly, slides were thawed and blocked with goat serum 5% (Sigma-Aldrich) PBS-T 1 × solution. After washing, they were incubated overnight at 4° with anti-macrophage antibody and anti- CD206 (Alexa Fluor 488 anti-mouse F4/80 and Brilliant Violet 605 anti-mouse CD206 Biolegend). Excess of the anti-neutrophil antibody was washed out with PBS 1X. Cells nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Slides were sealed with ProLong Gold antifade reagent (Life Technologies). Images were captured with a Nikon Eclipse Ti Inverted Fluorescent Microscope.