Kit 9707

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.).

When the NPCs reached 60% confluence, the NPCs were used for immunofluorescence staining. After dewaxing and hydration, tissue sections were used for immunohistochemical staining. Antigen repair, peroxidase elimination and serum blocking were performed according to the instructions. Specific primary antibodies were added and incubated at 4°C overnight. The NPC specimens were incubated with the fluorescently labeled secondary antibody at 37°C for 60 minutes. The tissue sections were also incubated with a special secondary antibody (KIT‐9707, MXB, China). Color development of tissue sections was performed using a DAB reagent. After the nuclei were stained with DAPI or hematoxylin (G1080, Solarbio, China), images of the specimens were captured using a microscope (Olympus) or laser confocal microscope (Lexia, Japan).