Chondrocyte Morphology and Collagen II Identification

Chondrocyte morphology on different culture days and of different generations was observed under an inverted phase-contrast microscope (Leica Microsystems, Inc., Wetzlar, Germany) and images were captured. Second-generation chondrocytes are often selected for experimentation (27 (link)); therefore, type II collagen immunohistochemistry was applied to identify passage 2 chondrocytes. A total of 5×10⁴ second-generation chondrocytes per well were implanted onto a sterile round coverglass in a 6-well plate. Chondrocytes in the 6-well plate (2 ml medium/well) were incubated for 48 h and were then randomly divided into two groups. The positive group was treated with 100 µl rabbit polyclonal antibody against collagen II (dilution 1:200; cat. no. ab34712; Abcam, Cambridge, UK), whereas the negative group was treated with 100 µl PBS. Both groups were incubated overnight at 4°C. After incubation, the two groups of chondrocytes were treated with a secondary antibody (cat. no. KIT-9707; MXB Biotechnologies, Inc., Fujian, China) at 37°C for 1 h, in accordance with the manufacturer's instructions; color was developed using a DAB kit (cat. no. DAB-0031; MXB Biotechnologies, Inc.); and the cells were stained with hematoxylin (Sigma-Aldrich; Merck KGaA) for 1 min. The staining of the two groups of cells was observed and compared under a phase-contrast microscope (Leica Microsystems, Inc.).