ABCD_AU543 recognizes collagen type II in cell-produced extracellular matrix by immunofluorescence

Animal-origin-free (AOF) antibodies are key to advancing ethical and reproducible in vitro research. In this study, we evaluated the recombinant AOF antibody ABCD_AU543 for the detection of collagen type II by immunofluorescence in cell-produced extracellular matrix (ECM). Collagen type II–rich ECM was generated using a CRISPRa-edited human adipose stem cell line overexpressing ACAN, COL2A1, and ZNF865. ABCD_AU543 specifically detected collagen type II using an animal-derived staining protocol, with staining patterns comparable to those previously reported. However, an entirely AOF staining protocol failed to detect collagen type II, despite successful cytoskeletal control staining. These results confirm the specificity of ABCD_AU543 for collagen type II and indicate that further optimization is required for fully animal-origin-free ECM immunostaining.