Enhanced dab kit

Adipose tissue obtained was fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into 4-mm-thick sections. IHC was performed according to the Vecta Stain Elite ABC kit protocols (Vector Laboratories, Burlingame, CA, USA). Antibody staining was visualized with the enhanced DAB kit (Vector Laboratories). The brown reaction product was quantified using Image-Pro Plus software. For IF staining, mature adipocytes were fixed in 4% paraformaldehyde for 15 min, pre-incubated in blocking buffer (10% normal donkey serum in phosphate-buffered saline (PBS)) for 30 min at room temperature, and incubated sequentially with primary and secondary antibodies diluted in blocking buffer. After washing with PBS, the samples were counterstained with DAPI. Cells were placed on slides using cover slips and mounting medium (SouthernBiotech, Burlingame, CA, USA), and examined under a fluorescence microscope (Nikon, Tokyo, Japan).

IHC was performed according to the Vectastain Elite ABC kit protocols (Vector Laboratories, Burlingame, CA, USA), and antibody staining was visualized with the enhanced DAB kit (Vector Laboratories). The brown reaction product was quantified using Image‐Pro Plus software.