Biotinylated rabbit anti rat secondary antibody

EAT was fixed in phosphate buffered 4% paraformaldehyde and embedded in paraffin. For immunohistochemistry, deparaffinized sections were rehydrated, and endogenous peroxide was inactivated by 3% H₂O₂. To reduce nonspecific staining, the sections were incubated in Block Ace (DS Pharma Biomedical Co., Osaka, Japan). After incubation with anti‐mouse Mac2 antibody (Cedarlane Laboratories, Ontario, Canada), sections were treated with biotinylated rabbit anti‐rat secondary antibody (Vector Laboratories, CA, USA) and then avidin–biotin complex (Vector Laboratories). The reaction products were visualized using diaminobenzidine and counterstained with hematoxylin.

Macrophages infiltrating WAT arrange around dead adipocytes, forming crown-like structures (CLS) [14 (link)], therefore we stained WAT for macrophage marker F4/80. Adipose tissue was fixed in 4% formaldehyde, embedded in paraffin and sectioned. Sections were then deparaffinized and rehydrated. Antigen retrieval was performed by incubation in citrate buffer, pH 6.0, for 15 minutes, and endogenous biotin was blocked using avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) for 15 minutes. Endogenous peroxidase activity was quenched by 30 minutes incubation in 0.6% hydrogen peroxide. Staining was performed using a primary F4/80 rat anti-mouse antibody (1:20, AbD Serotec, Raleigh, NC, USA) followed by a biotinylated rabbit anti-rat secondary antibody (1:200, Vector Laboratories). Binding of secondary antibody was visualized using an avidin biotinylated-horseradish peroxidase complex (Vector Laboratories) followed by DAB staining (Dako, Glostrup, Denmark). Sections were counterstained with Mayers hematoxylin. Images were obtained with a MIRAX Scan (Carl Zeiss, Göttingen, Germany) and analyses were done using BioPix iQ software (version 2.1.4., BioPix, Göteborg, Sweden). Representative micrographs were captured with an Olympus BX60F5 microscope with a 10X objective, connected to an Olympus DP72 camera.