Mouse anti human cd68 monoclonal antibody

To evaluate the structure and extracellular matrix (ECM) deposition of the regenerated cartilage, hematoxylin, and eosin (HE), Safranin-O, and type II collagen (mouse anti-human type II collagen monoclonal antibody, 1:100, Santa Cruz Biotechnology, Dallas, TX), staining was performed (Liu et al., 2008 (link)). The terminal deoxynucleotidyl transferase biotin dUTP nick end labeling assay (TUNEL) for apoptosis was analyzed using a TUNEL kit (Roche, Indianapolis, IN). CD3 was analyzed using mouse anti-human CD3 monoclonal antibody (1:200 in PBS, Santa Cruz Biotechnology, Santa Cruz, CA, United States). CD68 was analyzed using mouse anti-human CD68 monoclonal antibody (1:200 in PBS, Santa Cruz). Horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:200 in PBS, Santa Cruz) was then applied as a secondary antibody.

All the constructs targeted for histological analysis were bisected post gross evaluation. One-half of each construct was stored at −80°C for biochemical assays. The other half was fixed with 4% paraformaldehyde (fixing time: 24 h), embedded in paraffin, sectioned into slices (diameter: 5 μm), and mounted on glass slides. Sections were stained with hematoxylin and eosin (H&E) or Safranin O following standard protocols. Type II collagen, CD3, and CD68 were detected following the principles of immunohistochemistry. Mouse anti-human type II collagen monoclonal antibody (1:200 in PBS, Santa Cruz, CA, United States), mouse anti-human CD3 monoclonal antibody (1:200 in PBS, Santa Cruz, CA, United States), and mouse anti-human CD68 monoclonal antibody (1:200 in PBS, Santa Cruz, CA, United States) were used to study all the samples. Horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:200 in PBS, Santa Cruz, CA, United States) was then applied as a secondary antibody. Color development was achieved using diaminobenzidine tetrahydrochloride (DAB, Santa Cruz, CA, United States), and cell nuclei were counterstained with hematoxylin according to previously established techniques (Liu et al., 2016 (link)).