Histological Analysis of Liver and Adipose

Fresh liver and adipose tissue slices were fixed in 10% neutral-buffered formalin for 24 h, followed by paraffin embedding. Five µm sections were stained with hematoxylin and eosin (HE) to visualize tissue architecture and lipid droplets, as previously described [35 (link)]. Immunohistochemistry for F4/80 was performed using the avidin–biotin complex method. Tissue sections were deparaffinized, boiled in citrate buffer for antigen retrieval, and incubated with a primary antibody against F4/80 (Abcam, Cambridge, MA, USA), followed by biotinylated anti-rat IgG secondary antibody (Vector Biolabs, Burlingame, CA, USA). F4/80 was detected using 3,3’Diaminobenzidine (DAB) as a substrate, counterstained with hematoxylin, and imaged by brightfield microscopy. Negative controls were incubated without primary antibody.