Morphew Mary K, Giddings Thomas H, McIntosh J Richard
Laboratory for 3D Electron Microscopy, University of Colorado, Boulder, Colorado 80309-0347.
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347.
Cold Spring Harb Protoc. 2017 Jan 3;2017(1):2017/1/pdb.prot091322. doi: 10.1101/pdb.prot091322.
Electron microscopy (EM) immunolocalization of antigens in fission yeast can be accomplished with cells processed by rapid freezing and freeze-substitution followed by embedding in acrylic or methacrylate resins. Microtome sections of embedded cells are collected onto EM grids. Primary antibodies to the antigen of interest, followed by secondary antibodies conjugated to colloidal gold, are allowed to bind to antigens at the surface of these plastic sections. This type of postembed labeling provides information on antigen localization to a resolution of 10-20 nm, depending on the size of the metal particle used, the form of the antibody (Fab vs. complete IgG or IgM), and whether direct or indirect labeling is used. The method has the potential to map macromolecules in three dimensions in a relatively large volume when thin (30-60-nm) serial sections are labeled, imaged, aligned, and modeled to create a representative volume. The biggest challenge of this technique is the necessary compromise between the preservation of cellular ultrastructure and the preservation of antigen reactivity. The protocols described here show how to immunolabel samples for EM and include suggestions for overcoming challenges related to antigen preservation.
在裂殖酵母中，抗原的电子显微镜（EM）免疫定位可以通过对细胞进行快速冷冻和冷冻置换处理，然后包埋在丙烯酸或甲基丙烯酸树脂中来实现。将包埋细胞的切片收集到EM载网上。使针对目标抗原的一抗，随后是与胶体金偶联的二抗，与这些塑料切片表面的抗原结合。这种包埋后标记类型可提供抗原定位信息，分辨率可达10 - 20纳米，这取决于所使用金属颗粒的大小、抗体的形式（Fab片段与完整IgG或IgM）以及是否使用直接或间接标记。当对薄的（30 - 60纳米）连续切片进行标记、成像、对齐和建模以创建一个代表性体积时，该方法有潜力在相对较大的体积中对大分子进行三维映射。该技术最大的挑战在于细胞超微结构的保存与抗原反应性的保存之间的必要权衡。这里描述的方案展示了如何对用于EM的样本进行免疫标记，并包括克服与抗原保存相关挑战的建议。
