Oil red o staining

Samples were fixed for 20 h in Mildform 10N (Wako), embedded in paraffin, and then subjected to hematoxylin and eosin staining (Meyer’s hematoxylin solution and 1% eosin, Muto Pure Chemicals Co., Ltd., Tokyo, Japan) as well as immunohistochemistry using primary antibodies against F4/80 (Cedarlane Laboratories, Ontario, Canada). Oil Red O staining (Nacalai Tesque) was performed on frozen sections. For the image analysis, a microscope BZ-X710 (Keyence, Osaka, Japan), BZ-X Viewer version 1.3.1.1 (Keyence), and BZ-X Analyzer version 1.3.1.1 (Keyence) were used.

Intracellular lipid accumulation was detected using oil-red O staining (Nacalai Tesque). The adipocytes were rinsed with phosphate-buffered saline (PBS) twice and fixed in 10% buffered formalin for 10 min at room temperature. After rinsing with PBS twice, the cells were incubated with 60% isopropanol for 1 min. The staining started with oil-red O solution (oil-red O-saturated solution in isopropanol: water at 3:2) for 20 min. The cells were then washed with 60% isopropanol to remove background staining and finally rinsed with PBS. The stained cells were observed by microscopy.