Immunogold Labeling for Electron Microscopy: Strategy and Problem Solving
Immunogold labeling of antigens is a powerful technique in localizing antigens at the ultrastructural level. However, successful preservation of ultrastructure and antigens, and proper labeling of targets are always challenging. Sample processing using chemical fixation or cryofixation in conjunction with resin embedding, and cryo-thin sectioning techniques (Tokuyasu) are common methods used in immunogold labeling for electron microscopy. Chemical fixation and London resin (LR) White embedding are relatively easy methods suitable for on-section immunogold labeling and general ultrastructure preservation of most plant specimens. Cryofixation and LR White or Lowicryl embedding are optimal for both ultrastructure preservation and immunolabeling, but the need for expensive equipment limits their applications. The Tokuyasu method of cryo-thin sectioning is a very sensitive technique for immunolabeling but the process is more complicated and handling of specimen requires more expertise. Each labeling method has its merits and shortcomings. In this chapter, common problems and trouble-shooting procedures are addressed, and three common approaches for immunolabeling are presented.
KeywordsCryofixation–freeze substitution Formaldehyde Glutaraldehyde Immunogold Immunoelectron microscopy Tokuyasu method
We are especially grateful to Prof. Edward Yeung at University of Calgary, for his instruction and critical reading of this chapter. We also thank Tari Parmely and Dorothy Stanley at Stowers Institute for revising the English of the manuscript.
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