Mac 2

Immunohistochemical staining was performed as previously described [22 (link)] using primary antibodies against ANGPTL8 (1:200 dilution; Abcam, Cambridge, UK), α-smooth muscle actin (α-SMA) (1:200 dilution; ZM0003, ZSGB-BIO, Beijing, China), or galectin 2 (MAC-2) (1:200 dilution; Abcam, Cambridge, UK), followed by staining with secondary antibodies. Images were obtained with a Ni-UNikon Upright Microscope equipped with a DS-Ri2 color CCD (Nikon, Tokyo, Japan).

All tissue sections were first routinely examined by Hematoxylin and Eosin (H&E) staining to outline the wound and healing areas. Serial sections were deparaffinized and hydrated, followed by antigen retrieval by boiling in 10 mM citrate buffer (pH 6.0) using a microwave oven for 3 min at the highest power setting. Sections were allowed to cool at room temperature, washed twice in PBS, and treated with 5% hydrogen peroxide in methanol for 5 min to block the endogenous peroxidase activity. Following a PBS wash, skimmed milk powder (5%) was used to block non-specific background staining for 1 h. Sections were then incubated overnight at 4 °C with primary antibodies, namely TGF-β1 (Promega, 1:100), phospho-Smad2/3 (Cell signaling technology, 1:50), α-SMA, F4/80 and Mac-2 (All Abcam, 1:100). This was followed by incubation with horseradish peroxidase detection system (Biogenex, USA) for 20 min each and DAB (Sigma-Aldrich, USA) as the chromogenic substrate for visualization. Sections were imaged on a light microscope with a digital camera (Nikon).