Immunohistochemistry of types I and II collagen in undecalcified skeletal tissues

Types I and II collagen were demonstrated in semithin sections of undecalcified human endochondral growth plate, articular cartilage, and subchondral bone. The effects of several different methods for fixation, embedding, exposing of antigenic determinants, and immunoperoxidase staining were examined. Fixation in buffered formalin and paraformaldehyde-lysine-periodate solution gave more intense staining for collagens than fixation in paraformaldehyde-gluaraldehyde or Bouin's solution. Specimens embedded in Spurr epoxy resin yielded intense and uniform staining of areas known to contain the particular collagens after the resin had been removed by sodium ethoxide. The staining was enchanced following enzymatic digestion, especially with protease V (Sigma). Staining sensitivity and specificity were comparable with the indirect conjugate and double peroxidase-antiperoxidase (PAP) techniques; the PAP method was less sensitive. Embedment in methacrylate resins proved unsatisfactory because of exaggerated immunostaining of mineralized sites in comparison to unmineralized areas of the same tissues. In the growth plate specimens, type I collagen was identified in the matrices of bone, periosteum, perichondrium, and in the cytoplasm of hypertrophic and degenerative chondrocytes. Type II collagen was found uniformly throughout the cartilage matrix and in spicules of unresorbed cartilage matrix located in subchondral bone. A similar staining pattern was observed for the articular cartilage, except that type I collagen was not detected in chondrocytes.