Avidin biotin blocking kit

Cytospin samples from murine WT^tg, BAC-hCD83-2^tg, and BAC-hCD83-77^tg BMDCs were made by cytocentrifugation (Cytospin3; Shandon; Medite GmbH) of 200 μL fluid (2 × 10⁴ cells) at 1,000 rpm for 10 min on a Superfrost plus slide (Thermo Scientific, Braunschweig, Germany). Samples from human DCs were prepared as controls. Cytospins were air dried overnight and fixed in aceton for 3 min at −20°C. Immunohistochemistry was performed by using an avidin-biotin peroxidase detection system (Vector ABC Elite Kit, Vector laboratories, Burlingame, CA) according to manufacturer's protocol. Endogenous peroxidase activity was inhibited with 3% H₂O₂ and blocked with Avidin/Biotin Blocking Kit (Dako, Glostrup, Denmark) before addition of biotinylated anti-hCD83 (clone HB15e, Biolegend). Sections were counterstained with Mayer Hämalaun and mounted in Aquatex (Merck, Darmstadt, Germany). Images were observed using the light microscope Nikon Eclipse Ci.

For histological examination, a portion of the adipose tissue was fixed with 3.7% neutrally buffered formaldehyde and embedded in paraffin. The paraffin-embedded sections were deparaffinized and incubated for 30 min with 0.3% H₂O₂ in methanol to block endogenous peroxidase. Endogenous avidin and biotin were blocked using an Avidin-Biotin Blocking kit (DAKO, Carpentaria, CA, USA), as described previously³⁰ . Tissue sections were incubated overnight at 4 °C with primary antibody against Ucp1 (U6382, 1:50; Sigma), followed by 1 h at room temperature with peroxidase-conjugated anti-rabbit IgG secondary antibody (Histofine Simple Stain Max-PO (R); 1:1000; Nichirei, Tokyo, Japan). Immune complexes were visualized using the peroxidase stain DAB kit (brown stain; Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s instructions. Confocal microscopic imaging was used to visualize results.