title: The detection of glycosaminoglycans in pancreatic islets and lymphoid tissues.
abstract: In this chapter, we describe the detection of the glycosaminoglycans hyaluronan and heparan sulfate in pancreatic islets and lymphoid tissues. The identification of hyaluronan in tissues is achieved by utilizing a highly specific hyaluronan binding protein (HABP) probe that interacts with hyaluronan in tissue sections. The HABP probe is prepared by enzymatic digestion of the chondroitin sulfate proteoglycan aggrecan which is present in bovine nasal cartilage, and is then biotinylated in the presence of bound hyaluronan and the link protein. Hyaluronan is then removed by gel filtration chromatography. The biotinylated HABP-link protein complex is applied to tissue sections and binding of the complex to tissue hyaluronan is visualized by enzymatic precipitation of chromogenic substrates. To determine hyaluronan content in tissues, tissues are first proteolytically digested to release hyaluronan from the macromolecular complexes that this molecule forms with other extracellular matrix constituents. Digested tissue is then incubated with HABP. The hyaluronan-HABP complexes are extracted and the hyaluronan concentration in the tissue is determined using an ELISA-like assay. Heparan sulfate is identified in mouse tissues by Alcian blue histochemistry and indirect immunohistochemistry. In human tissues, heparan sulfate is best detected by indirect immunohistochemistry using a specific anti-heparan sulfate monoclonal antibody. A biotinylated secondary antibody is then applied in conjunction with streptavidin-peroxidase and its binding to the anti-heparan sulfate antibody is visualized by enzymatic precipitation of chromogenic substrates.
authors.affiliation: Matrix Biology Program, Benaroya Research Institute at Virginia Mason, 1201 Ninth Avenue, Seattle, WA, 98101, USA.
authors.forename: Marika
authors.initials: M
authors.lastname: Bogdani
authors.forename: Charmaine
authors.initials: C
authors.lastname: Simeonovic
authors.forename: Nadine
authors.initials: N
authors.lastname: Nagy
authors.forename: Pamela Y
authors.initials: PY
authors.lastname: Johnson
authors.forename: Christina K
authors.initials: CK
authors.lastname: Chan
authors.forename: Thomas N
authors.initials: TN
authors.lastname: Wight
mesh_terms: D000818:Animals; D019921:Biotinylation; D002417:Cattle; D002850:Chromatography, Gel; D006025:Glycosaminoglycans / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000378:metabolism*; D008221:Lymphoid Tissue / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight
chemical_list: D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid