2.7. Staining of tissue sections

EWAT, liver, pancreas, and intestinal were harvested and fixed overnight in 4% paraformaldehyde, paraffin-embedded, then sectioned (5 μm), followed by hematoxylin and eosin (H&E) staining. Adipose tissue sections were hybridized with CD3 (Cat#AF20162, AiFang, Changsha, China) and F4/80 (Cat#SAF002, AiFang, Changsha, China) antibodies. Alexa Flour 488 donkey anti-mouse IgG (Cat# A32766, Invitrogen, USA) was used as a secondary antibody to detect CD3⁺ cells. Alexa Fluor 594 goat anti-rabbit IgG (Cat#A32740, Invitrogen, USA) was used as a secondary antibody to detect F4/80⁺ cells. For the detection of macrophage apoptosis, the TUNEL assay was performed using a FITC TUNEL cell apoptosis detection kit (Cat# G1501-100T, Servicebio, Wuhan, China).

Pancreatic paraffin-embedded tissue sections were stained with mouse anti-insulin (Cat# 66198-1, Proteintech, USA), rabbit anti-Glucagon (Cat#ab92517, Abcam, The UK), rabbit anti-Ki67 (Cat# D3B5, Cell Signaling Technology, USA) antibodies. Alexa Flour 488 donkey anti-mouse IgG (Cat# A32766, Invitrogen, USA) and Alexa Fluor 594 goat anti-rabbit IgG (Cat# A32740, Invitrogen, USA) were used as secondary antibodies. Intestinal paraffin-embedded tissue sections were stained with mouse anti-GLP1 (Cat# sc-514592, Santa Cruz, USA). Images were acquired with an Olympus microscope and integrated density was analyzed with Image J Software.