Af5202

To test whether IP-HT grafts could influence the phenotype of macrophages, we carried out immunohistochemistry according to the previously reported standard method.^{6 (link)}
The tendon-to-bone interface at a depth of 5 mm from the tibial joint surface was evaluated. After deparaffinzing and rehydrating, the paraffin section underwent antigen retrieval. Then 3% hydrogen peroxide was used to block endogenous peroxidase activity. After serum sealing, primary and secondary antibody incubations, DAB chromogenic reaction, and nucleus counterstaining, the section was dehydrated and mounted. According to the results of antibody activity in our preliminary experiment, anti-CD206 (DF7523; Affinity), anti-iNOS (DF7688; Affinity), anti–TNF-α (AF5202; Affinity), and anti–TGF-β (AF6330; Affinity) were the primary antibodies utilized. According to a previous report,^{31 (link)}
the positive area at the tendon-to-bone interface was quantified (the percentage of positive cells) using ImageJ software (US National Institutes of Health) by the third author (Z.L.), who was blind to group allocation. Three slices of each sample were quantified to obtain an average value, representing the value of this sample.