Hrp coupled secondary antibodies

To assess blood vessel formation, immunohistochemistry was performed on day 6. The tissue sections described above were incubated with primary antibodies (anti-collagen type IV, 1 : 100, and anti-CD31, 1 : 100, Abcam, United Kingdom) overnight at 4°C and then incubated with HRP-coupled secondary antibodies (Aspen, China). Staining was performed using diaminobenzidine (brown). For each section, tube-like structures were considered to be newly formed blood vessels and were quantified. Furthermore, to quantify the number of mature blood vessels, six sections from different tissue samples were selected from each treatment group for immunofluorescence analysis on day 9. CD31 (for endothelial cells), α-SMA (for smooth muscle cells), and cell nuclei were stained red, green, and blue, respectively. Red and green costaining was considered to represent mature blood vessels. All of the evaluation procedures were performed independently by two pathologists.

Immunohistochemistry was performed to assess the deposition of collagen II in the chondrocytes, according to manufacturer's instructions. Briefly, the slides of cells or tissue sections described above were incubated with primary antibodies (anti-collagen type II, 1 : 150, and anti-CD31, 1 : 100, Abcam, United Kingdom) overnight at 4°C and then incubated with HRP-coupled secondary antibodies (Aspen, China). Staining was performed using diaminobenzidine (brown).