GLYCAN DETECTION BY LECTIN HISTOCHEMISTRY

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： 采用生物素化凝集素-链霉亲和素过氧化物酶系统进行凝集素组织化学。简言之，将切片在二甲苯中脱蜡，分别在降级乙醇和蒸馏水中水合。用3% H2 O2水溶液处理切片30分钟，消除内源性过氧化物酶。然后用TBS（20 mM Tris-HCL，pH 7.4，含0.15 M NaCl、10 mM CaCl 2和1 mM MnCl 2）洗涤切片。通过用封闭溶液（由试剂盒提供）处理载玻片1 h来封闭非特异性染色。将TBS中的10 μ g /ml生物素化凝集素（表1，Vector，USA）应用于切片，并在室温下孵育1小时。用TBS洗涤30分钟后，将切片与Vectastain ABC试剂（Vector，USA）孵育30分钟。用TBS洗涤30分钟后，使用二氨基联苯胺（DAB）试剂进行显色。然后用Mayer苏木精复染切片，脱水，在二甲苯中澄清，并用DePex封固剂封固。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Lectin histochemistry was carried out using biotinylated lectin-streptavidin peroxidase system. Briefly, sections were deparaffinized in xylene, hydrated in downgraded ethanol and distilled water, respectively. The endogenous peroxidase was eliminated by treating sections with 3% H2O2 in water for 30 min. Sections were then washed in TBS (20 mM Tris-HCL, pH 7.4, containing 0.15 M NaCl, 10mM CaCl2 and 1mM MnCl2). Non-specific staining was blocked by treating slides with blocking solution (supplied by the kit) for 1 h. Biotinylated lectins (Table 1, Vector, USA) 10 ¿g /ml in TBS were applied to the sections and incubated for 1 h at room temperature. After washing with TBS for 30 min, the sections were incubated with Vectastain ABC reagent (Vector, USA) for 30 min. Color development using diaminobenzidine (DAB) reagent was done after washing with TBS for 30 min. Sections were then counterstained with Mayer's hematoxylin, dehydrated, cleared in xylene, and mounted with DePex mounting medium.