Visualization of Different Tissues Involved in Endochondral Ossification With Alcian Blue Hematoxylin and Orange G/Eosin Counterstain

The histologic evaluation of endochondral ossification is critical to the study of fracture healing, developmental biology, and comparative histology. A modification of Sayers’ alcian blue hematoxylin staining technique is described in which a different counterstain is applied. Paraffin sections are stained in alcian blue hematoxylin for 30 min, and then placed in orange G-eosin counterstain for 1 min, 30 s. This method can differentiate cartilage, mature bone, and immature bone found in various stages of endochondral ossification and fracture callus. Increasing the hematoxylin content to 0.5% produced a more striking contrast between the bone and cartilaginous aspects of the healing fracture callus. Inclusion of orange G (instead of acid fuchsin) in the counterstain provides better demarcation of mature and immature bone. Alcian blue hematoxylin/orange G-eosin consistently stains cartilage blue, mature bone orange, and immature bone mauve and is particularly suited to the study of endochondral ossification, fracture healing, and bone remodeling. (The J Histotechnol 31:19, 2008) Submitted May 29, 2007; accepted with revisions October 1, 2007