GLYCAN DETECTION BY LECTIN HISTOCHEMISTRY

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： 用生物素标记的凝集素-链霉亲和素过氧化物酶系统进行凝集素组织化学研究。简而言之，切片在二甲苯中脱蜡，分别用降级乙醇和蒸馏水水合。用3%的双氧水处理切片30min，可清除内源过氧化物酶。切片在TBS(20 mM Tris-HCl，pH 7.4，含0.15 M氯化钠、10 mM CaCl2和1 mM MnCl2)中洗涤。用试剂盒提供的封闭液处理玻片1h，阻断非特异性染色，将生物素化凝集素(表1，矢量，美国)10g/mlTBS涂于切片上，室温孵育1h。用TBS洗涤30min后，用Vectaain ABC试剂(美国Vectaain ABC试剂)孵育30min。用TBS洗涤30min后，用二氨基联胺(DAB)试剂显色。切片用Mayer‘s苏木精反染，脱水，在二甲苯中清除，并用DePex安装介质固定。

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.