Anti f4 80 antibody

Fresh BAT, epididymal WAT, liver, and qM tissues were fixed in 10% (v/v) formalin, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin (H&E) or immunohistochemically using anti-F4/80 antibody (#70076; Cell Signaling Technology, Danvers, USA) and anti-UCP1 antibody (sc-518024, Santa Cruz Biotechnology, Santa Cruz, USA)
[24] (link). In brief, the sections were heated (65°C, 2 h), dewaxed, rehydrated, and boiled (2 min) in sodium citrate (10 mM, pH6.0) for antigen retrieval. The slides were then treated with 3% H
₂O
₂ for 30 min to eliminate endogenous peroxidase activity and blocked with 5% BSA for 1 h. The slides were incubated with primary antibody at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody (A0208; Beyotime, Shanghai, China) for 1 h at room temperature. Color development was performed by incubation with DAB reagent (P0203; Beyotime), and the slides were counterstained with hematoxylin according to manufacturer’s protocols.

Portions of fresh liver were formalin-fixed and paraffin-embedded and subsequently subjected to hematoxylin and eosin (H&E) and Sirius red staining. IHC was conducted as previously described. Briefly, paraffin-embedded liver sections were deparaffinized and rehydrated, followed by incubation with anti-F4/80 antibody (Cell Signaling Technology, Danvers, MA, USA). For Oil red O staining, frozen liver portions were stained with Oil red O dye (Sigma, St. Louis, MO, USA) according to the manufacturer’s protocols. Three independent visual fields were randomly selected from each slide at 20× magnification and analyzed using ImageJ software. Pathological features were estimated according to the NAFLD activity score (NAS), Sirius red area, Oil red O staining area, and F4/80 staining intensity.