Abstract
Immunostaining is the process of identifying proteins in tissue sections by incubating the sample with antibodies specific to the protein of interest, then visualizing the bound antibody using a chromogen (immunohistochemistry or IHC) or fluorescence (immunofluorescence or IF). Unlike in situ hybridization, which identifies gene transcripts in cells, immunostaining identifies the products themselves and provides information about their localization within cells (nuclear, cytoplasmic, or membrane) or extracellular matrix. This can be particularly important in the context of bone and cartilage because they contain many cell types as well as matrix components, each with distinct protein expression patterns. As the number of antibodies continues to grow, this technique has become vital for research laboratories studying the skeleton. Here, we describe a detailed protocol for antibody-based in situ analysis of bone and associated tissues, addressing specific issues associated with staining of hard and matrix-rich tissues.