Protein Localization in Wax-Embedded and Frozen Sections of Bone Using Immunohistochemistry

Abstract

Immunohistochemistry can provide valuable information regarding protein expression in different cell types at specific stages of differentiation during bone modeling and remodeling. By combining immunohistochemistry with other techniques, it is possible for the researcher to determine protein expression, in relation to mRNA production, enzyme activity and bone remodeling, on the same sample of bone. This chapter covers the localization of protein in human bone by immunohistochemistry using an indirect immunoperoxidase method, and considers both frozen and wax-embedded sections. Methods for immunostaining of plastic embedded tissue can be found in the chapter by Van Leeuwen and Derkx, this volume. Immunohistochemistry is based on incubating high-affinity antibodies on tissue sections to detect patterns of expression for specific antigens within the tissue. This can be achieved using either an immunofluorescence-based technique in which the antibody is conjugated with a fluorochrome, or an enzyme-labeled antibody method. The latter procedure has several advantages; it utilizes the same enzyme complexes for all primary antisera irrespective of its origin in different animal species; stained sections may be permanently mounted; and sections can be viewed using brightfield microscopy. Detection methods have increased the sensitivity of immunohistochemistry. There are now several ways that the signal of antibody-antigen binding can be amplified, thus allowing the use of only small amounts of antibody.