Matrix Biology Program, Benaroya Research Institute at Virginia Mason, Seattle, WA, USA.
Diabetes/Transplantation Immunobiology Laboratory, Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.
Methods Mol Biol. 2022;2303:695-717. doi: 10.1007/978-1-0716-1398-6_53.
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.Historically, heparan sulfate was identified in tissue sections using the cationic dye Alcian blue and histochemistry based on the critical electrolyte concentration principle of differential staining of glycosaminoglycans using salt solutions. For both human and mouse pancreas sections, the current optimal method for detecting heparan sulfate is by indirect immunohistochemistry using a specific anti-heparan sulfate monoclonal antibody. A peroxidase-conjugated secondary antibody is then applied, and its binding to the anti-heparan sulfate antibody is visualized by oxidation and precipitation of a chromogenic substrate.
在本章中，我们描述了对胰岛和淋巴组织中的糖胺聚糖透明质酸和硫酸乙酰肝素的检测。组织中透明质酸的鉴定是通过利用与组织切片中的透明质酸相互作用的高度特异性透明质酸结合蛋白（HABP）探针来实现的。HABP 探针通过在存在结合的透明质酸和链接蛋白的情况下用酶消化存在于牛鼻软骨中的软骨素硫酸盐蛋白聚糖聚集素来制备，并在该结合物中进行生物素化。然后通过凝胶过滤色谱法去除透明质酸。将生物素化的 HABP-链接蛋白复合物应用于组织切片，通过酶促沉淀显色底物来可视化复合物与组织透明质酸的结合。为了确定组织中的透明质酸含量，首先通过蛋白水解酶消化将透明质酸从该分子与其他细胞外基质成分形成的大分子复合物中释放出来。然后将消化的组织与 HABP 孵育。提取透明质酸-HABP 复合物，并使用 ELISA 样测定法测定组织中透明质酸的浓度。历史上，使用阳离子染料阿利新蓝和基于盐溶液对糖胺聚糖进行差异染色的临界电解质浓度原理的组织化学法在组织切片中鉴定硫酸乙酰肝素。对于人胰腺和小鼠胰腺切片，目前检测硫酸乙酰肝素的最佳方法是通过使用特异性抗硫酸乙酰肝素单克隆抗体的间接免疫组织化学法。然后应用过氧化物酶缀合的二抗，并且通过氧化和显色底物沉淀来可视化其与抗硫酸乙酰肝素抗体的结合。
