期刊
TISSUE & CELL
卷 57, 期 -, 页码 90-102出版社
CHURCHILL LIVINGSTONE
DOI: 10.1016/j.tice.2018.08.010
关键词
Immunoelectron microscopy; Cryo-ultramicrotomy; Postembedding labeling; Ultrastructure; Correlative light and electron microscopy (CLEM); Label quantification
资金
- DFG Research Center Nanoscale Microscopy and the Molecular Physiology of the Brain [EXC171]
- Cluster of Excellence
Since the pioneering work of Kiyoteru Tokuyasu in the 70ths the use of thawed cryosections prepared according to the Tokuyasu-method for immunoelectron microscopy did not lose popularity. We owe this method a whole subcellular world described by discrete gold particles pointing at cargo, receptors and organelle markers on delicate images of the inner life of a cell. Here we explain the procedure of sample preparation, sectioning and immunolabeling in view of recent developments and the reasoning behind protocols including some historical perspective. Cryosections are prepared from chemically fixed and sucrose infiltrated samples and labeled with affinity probes and electron dense markers. These sections are ideal substrates for immunolabeling, since antigens are not exposed to organic solvent dehydration or masked by resin. Instead, the structures remain fully hydrated throughout the labeling procedure. Furthermore, target molecules inside dense intercellular structural elements, cells and organelles are accessible to antibodies from the section surface. For the validation of antibody specificity several approaches are recommended including knock-out tissue and reagent controls. Correlative light and electron microscopy strategies involving correlative probes are possible as well as correlation of live imaging with the underlying ultrastructure. By applying stereology, gold labeling can be quantified and evaluated for specificity.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据