Immunohistochemistry for Protein Analysis

Immunohistochemical staining was performed as previously described [44 (link)]. Tissue slices were deparaffinized and rehydrated. After that, microwave was used to retrieve the antigen. Next, we used 3% hydrogen peroxide to block the endogenous peroxidase activity and 5% BSA to block the non-specific binding sites under room temperature. The primary antibodies were incubated with slices overnight at 4 °C. The next day, an appropriate HRP-conjugated secondary antibody was used to incubate with sections and counterstained with hematoxylin. Immunohistochemistry was done using the following primary antibodies: NR2F2 (A10251; Abclonal), EGR1 (55,117–1-AP; Proteintech), SOX5 (A6985; Abclonal), COMP (A13963; Abclonal), Collagen X (ab49945; Abcam), POSTN (A14556; Abclonal), CD68(A13286; Abclonal), and CD31(ab9498; Abcam).