Blocking buffer

Sections (4 mm) from mouse lymph nodes and kidneys were treated with formalin and paraffin and further subjected to either haematoxylin and eosin (H&E) staining or immunohistochemistry (IHC) staining. For the latter, sections were further dewaxed with xylene, 95% ethanol and 70% ethanol. Primary and secondary antibodies were then sequentially applied at room temperature (RT) (30 min). The antibodies are listed in Table S6. Slides were analysed by a digital fluorescence microscope (Leica) and NLS Elements Basic Research Imaging Software (Leica). For multicolour IHC staining, an Opal Seven‐Color Kit (Perkin Elmer) was applied. Briefly, sections of human skin and tonsil tissues underwent epitope retrieval in citrate buffer (Perkin Elmer) and incubation with blocking buffer (Perkin Elmer) and then primary antibodies at RT (30 min). Background staining was further reduced, and sections were rinsed in citrate buffer. Sections were finally scanned by a Vectra imaging system (Perkin Elmer, Vectra 3.0.3) and analysed by inForm (Perkin Elmer, inForm 2.3.1).

Ears were separated, and only the part showing vasculature was kept and fixed in 4% PFA for 30 min. After three washes in PBS, nonspecific binding was blocked by incubation of the ears in 0.1 M Tris–HCl, 0.3 M NaCl, blocking buffer (Perkin Elmer) (pH 7.4) containing 0.5% Triton X-100 (TBNT) for 4 h at RT. The anti-TH antibody (Merck Millipore, Saint-Quentin-en-Yvelines, France, Cat# AB152) was then added and incubated overnight at 4 °C. Anti-α-SMA-Cy3 antibody (Sigma‒Aldrich, Cat# C6198) and donkey anti-rabbit IgG-AF647 (Invitrogen, Carlsbad, CA, USA, Cat# A-31573) were then incubated for 4 h after at least three washes with 0.1 M Tris–HCl, 0.3 M NaCl (pH 7.4) containing 0.5% Triton X-100 (TNT). Ears were mounted in Dako fluorescent medium (Agilent Technologies, Cat# S3023), and images were acquired with the Zeiss Axioimager Z2 Apotome (Carl Zeiss Microscopy, Jena, Germany). The antibodies used are listed in Supplementary Table S1.