Ulex europaeus agglutinin

All chemical reagents are from MilliporeSigma (Göttingen, Germany), unless stated otherwise. Recombinant proteins including murine (m)Dectin-1 (mDectin-1) and -2, human (h)Dectin-1 and -2 and DC-SIGN were purchased from R&D Systems (Abingdon, United Kingdom), murine SIGN-R1 was from Sino Biologic (Beijing, China). The NSO cell line was used for mDectin-1 and -1 and hDectin-1 and -2 recombinant production, CHO cell line was used for recombinant production DC-SIGN and human embryonic kidney cells for recombinant SIGN-R1. Antibodies used in the study include mouse monoclonal anti-Gal-3 (R&D Systems) and goat anti-mouse IgG horseradish peroxidase conjugate (MilliporeSigma). The following fluorescein labeled plant lectins, wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), and Ulex europaeus agglutinin (UEA) , were obtained from Vector Laboratories (Peterborough, United Kingdom), β-1,6-galactobiose (galactobiose) from Dextra Labs (Reading, United Kingdom), and scleroglucan from Elicity (Crolles, France). C3GnT^−/− mice were from Dr. Lijun Xia (University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA).

Keratinocyte cells were recovered at each time point (T0, 1 and 2 months) and permeabilized with PBS-Triton 0.1% for 4 min at 4 °C. Keratinocyte cells and fingermark samples were saturated in PBS-BSA 1% for 30 min at room temperature. Samples were incubated for 20 min in a humidity chamber using a biotinylated lectin solution (at final concentration 10 µg/ml): Concanavalin A (ConA), Peanut agglutinin (PNA), Elderberry lectin (SNA), and Ulex europaeus agglutinin (UEA) (B-1005, B-1075, B-1305, and B-1065 respectively, Vector Laboratories, USA). After washing with PBS, samples were incubated with Alexa Fluor 555 Streptavidin (S21381, Thermofisher, USA), diluted 1:600, for 20 min in a dark chamber. DNA revelation of the nuclei was done using DNA probes: DAPI or Hoechst 33342 both diluted 1:3000 in PBS-BSA 0.5%. With anti-histone H2B (SAB450223, Sigma-Aldrich, USA), samples were incubated for 2 h (diluted 1/250 in PBS-BSA 0.5%) and then they were incubated with the appropriate fluorescent secondary antibodies for 1 h in the dark (Alexa Fluor 488 conjugated anti-mouse antibody (A11029, Life Technologies, USA)).
Slides were mounted with Prolong mounting medium. Controls without primary antibodies were negative.

Three biotinylated fucose-specific lectins: Lotus tetragonolobus agglutinin (LTA, catalog No. B-1325, Vector Laboratories Inc., Burlingame, CA, USA), Ulex europaeus agglutinin (UEA, catalog No. B-1065, Vector Laboratories Inc., Burlingame, CA, USA) and Lens culinaris agglutinin (LCA, catalog No. B-1045, Vector Laboratories Inc., Burlingame, CA, USA) were used to determine fucose expression in the lectin-ELISA procedure according to the Kratz et al.²⁴ with modifications described below. The specificity of lectins is not absolute, and they can react with more than one oligosaccharide residue. Lotus tetragonolobus agglutinin and Ulex europaeus agglutinin detect fucoses linked to the galactose or antennary N-acetylglucosamine by α1,3 glycosidic bond and α1,2 glycosidic bond, respectively^{28 (link)}. Lens culinaris agglutinin was used for core fucose detection^{29 (link)}.