Vectastain abc kit reagents

Paraffin-embedded epididymal EWAT and liver sections (7 μm) were processed as previously described [10] (link). Briefly, slides were deparaffinized and processed for antigen retrieval with a 2100 Retriever using 13 DIVA solution and Hot Rinse (HistoLab Products AB, Gothenburg, Sweden). Endogenous peroxidase activity was quenched with 30% H₂O₂ for 30 min. Blocking in 5% rabbit serum, 1% bovine serum albumin, and 0.1% Triton X-100 at room temperature for 30 min. EWAT macrophages were stained with MAC-2/galectin-3 antibody (diluted 1:500 in blocking buffer overnight at 4 °C), and then detected with a biotinylated anti-rat (10 mg/ml) antibody. Immune complexes were detected by Vulcan Red reagent (Vector Laboratories, USA), according to the manufacturer's instructions. Crown-like structures were counted in 15–40 mm² of histological sections per tissue. For the liver staining, F4/80 antibody was diluted 1:500 and incubated in blocking buffer 2 h at room temperature and then detected with a HRP anti-rat antibody diluted 1:100. Immune complexes were detected by Vectastain ABC kit reagents (Vectorlab, USA) according to the manufacturer's instructions counterstained with hematoxylin and quantitated by densitometric analysis using Biopix iQ software (version 2.1.3; Biopix, Sweden). List of antibodies is reported in online Supplementary Table 3.