Immunohistochemistry (IHC) Staining and Analysis

A standard biotin-streptavidin horse radish peroxidase (HRP) detection system was used for visualization of immunostained protein. Paraffin-embedded tissues were first cut into 5-μm thick sections. Then, after a series of steps, including dewaxing and hydration, antigen repair, and blocking endogenous peroxidase, the sections were immunoreacted with an FABP4 rabbit monoclonal antibody (ab92501; Abcam, Cambridge, UK), by incubating overnight. Afterwards, the sections were washed with sterile phosphate-buffered saline (PBS) and incubated with rabbit anti-mouse biotin-bound secondary antibody. The FABP4-immunoreacted sections were observed under a microscope and interpreted by qualified pathologists, staining scoring criteria were as previously described.¹⁴ Tissue sections with a final staining score ≥3 were considered to be positive.