Primary antibody against f4 80

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.

The frozen sections were fixed in the ice-cold mixed liquor of acetone and methanol, dried, dehydrated, and blocked by 5% goat serum. The sections were incubated with the primary antibody against F4/80 (Abcam, Cambridge, MA, USA) at room temperature for 2 h. Then, the sections were incubated with Alexa Fluor Goat pAb to Rat IgG (Abcam, Cambridge, MA, USA) at room temperature for 1 h. Incubated with DAPI, the sections were visualized under microscope. The immunofluorescence staining was analyzed with Image Pro-Plus 6.0, expressed in MOD (Wan et al., 2010 (link)).

After routine deparaffinization, hydration, and blockage of endogenous peroxidase, sections were pretreated by microwaving for 20 min in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval. This was followed by incubation sequentially with a blocking agent, α‐SMA antibodies (1:100, Boster, China), and a secondary antibody (1:200, Promega, Madison, WI). Slides were incubated with diaminobenzidine, followed with a brief counterstaining with hematoxylin, and mounted with Vectashield (Vector Labs, Burlingame, CA). The areas‐of‐interest were photographed and converted to a digital image using a light microscope equipped with a camera (Olympus BX51, NY). For immunofluorescence staining, liver specimens were fixed in 10% buffered formalin and sequentially exposed to 10 and 30% sucrose in PBS for 10 hr each and then embedded in Tissue Tek OTC compound (Sakura Finetek, Torrance, CA). The liver sections were permeabilized using 0.25% Triton X‐100 and incubated with a primary antibody against F4/80 (1:200, Abcam, Cambridge, MA). The liver sections were then incubated with the corresponding Alexa Fluor 594‐conjugated secondary antibodies (1:200, Invitrogen, Carlsbad, CA) for 1 hr at room temperature and stained with DAPI (1 μg/ml) for 10 min. Finally, the stained sections were viewed and photographed using a confocal microscope (Leica TCS SP5, Leica, Mannheim, Germany).