Immunohistochemical comparison of differentiation markers on paraffin and plastic embedded human bone samples

To assess bone pathologies and bone regeneration immunohistochemistry may provide additional information compared to conventional histology. However, the effectiveness of this technique is limited due to tissue fixation, preparation and embedding. For bone tissue the standard immunohistological procedure includes formalin fixation, followed by decalcification and paraffin embedding. This may lead to a badly preserved trabecular bone structure but allows antibody application. Alternatively, methyl-methacrylate (MMA) resin may be used for embedding, thus circumventing the decalcification procedure. In this study immunohistology of typical bone markers was compared using human bone samples fixed either with alcohol or formalin and further decalcified and embedded in paraffin and decalcified or non decalcified samples embedded in Technovit 9100 New(R). On semi-thin sections immunohistochemistry with bone markers osteocalcin, osteonectin, osteopontin, collagen type I and the cellular markers CD34 and CD68 was performed. Independent of the fixative used, Technovit 9100 New embedded non-decalcified bone yielded a stronger immunostaining for all markers when compared to decalcified bone embedded either in methyl-methacrylate or paraffin. In addition there was a better preservation of the trabecular bone morphology. The immunohistochemical results demonstrate that Technovit 9100 New as a low-temperature acrylic resin embedding method can be favoured over paraffin embedding.