Skepper Jeremy N, Powell Janet M
CSH Protoc. 2008 Jun 1;2008:pdb.prot5015. doi: 10.1101/pdb.prot5015.
INTRODUCTIONIn post-embedding methods of immunogold staining, the cells or tissues are fixed chemically or cryoimmobilized, dehydrated, and embedded in epoxy or acrylic resins. Thin sections (50-70 nm in thickness) are cut using an ultramicrotome with a diamond knife, using a water bath to collect the sections as they slide off the knife. The sections are stretched with solvent vapor or a heat source and collected onto either bare or plastic-coated nickel grids. The sections are then stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections. The primary antibodies are visualized by staining immunochemically with secondary antibodies raised against the species and isotype of the primary antibodies, conjugated to colloidal gold particles. The immunochemically stained sections are then contrast stained with salts of uranium (uranyl acetate) and lead (lead citrate) to reveal the ultrastructure of the cells, and are finally viewed by transmission electron microscopy (TEM). Chemical fixation and embedding in a highly cross-linked epoxy resin is the method of choice for optimal ultrastructure and stability of the thin section in the electron beam. Immunogold staining of thin epoxy resin sections, described here, is useful if the antigen of interest is very resistant to fixative, or if only archived material that was fixed primarily for ultrastructural studies is available.
引言
在免疫金染色的包埋后方法中，细胞或组织通过化学固定或冷冻固定、脱水，然后包埋在环氧树脂或丙烯酸树脂中。使用配备金刚石刀的超薄切片机切出厚度为50 - 70纳米的薄片，切片从刀上滑落时，使用水浴收集。切片用溶剂蒸汽或热源拉伸，然后收集到裸镍网或塑料涂层镍网上。然后用针对切片表面暴露抗原产生的一抗进行免疫化学染色。通过用针对一抗的物种和同种型产生的二抗进行免疫化学染色来可视化一抗，二抗与胶体金颗粒偶联。然后将免疫化学染色的切片用铀盐（醋酸双氧铀）和铅盐（柠檬酸铅）进行对比染色，以揭示细胞的超微结构，最后通过透射电子显微镜（TEM）观察。化学固定并包埋在高度交联的环氧树脂中是获得最佳超微结构和薄片在电子束中稳定性的首选方法。本文所述的环氧树脂薄片的免疫金染色，在感兴趣的抗原对固定剂非常耐受，或者仅可获得主要为超微结构研究而固定的存档材料时很有用。
