Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Griffiths G (1993) Fine structure immunocytochemistry. Springer-Verlag, Berlin
Tokuyasu KT (1973) A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 57:551–565
Tokuyasu KT (1986) Application of cryoultramicrotomy to immunocytochemistry. J Microsc 143:139–149
Kiss JZ, McDonald K (1993) Electron microscopy immunocytochemistry flowing cryofixation and freeze substitution. Meth Cell Biol 37:311–341
Newman GR, Hobot JA (1987) Modern acrylics for post-embedding immunostaining techniques. J Histochem Cytochem 35:971–981
Newman GR, Hobot JA (2001) Resin microscopy and on-section immunocytochemistry. Springer–Verlag, New York
McDonald K (1999) High-pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabeling. Meth Mol Biol 117:77–97
McDonald K (2007) Cryopreparation methods for electron microscopy of selected model systems. Meth Cell Biol 79:23–56
McDonald K (2014) Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material. Protoplasma 251:429–448
Takeuchi M, Takabe K, Momeyuki Y (2010) Immunoelectron microscopy of cryofixed and freeze substituted plant tissues. Meth Mol Biol 657:155–165
Karahara I, Kang B-H (2014) High-pressure freezing and low-temperature processing of plant tissue samples for electron microscopy. In: Žárský V, Cvrčková F (eds) Plant cell morphogenesis, vol 1080. Methods in molecular biology. Humana Press, New York, pp 147–157
Vaughn KC, Campbell WH (1988) Immunogold localization of nitrate reductase in maize leaves. Plant Physiol 88:1354–1357
Kuo J (2007) Processing tissues for ultrastructural study in electron microscopy: methods and protocols. Humana Press, New York
Hayat MA (2000) Principles and techniques of electron microscopy: biological applications, 4th edn. Cambridge University Press, New York
Ripper D, Schwarzt H, Stierhof YD (2008) Cryo-section immunolabeling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration. Biol Cell 100:109–123
Waguri S, Kohmura M, Gotow T, Watanabe T et al (1999) The induction of autophagic vacuoles and the unique endocytic compartments, C-shaped multivesicular bodies, in GH4C1 cells after treatment with 17beta-estradiol, insulin and EGF. Arch Histo Cytol 62:423–434
Dettmer J, Hong-Hersdorf A, Stierholf Y-D, Schumacher K (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18:715–730
Slot J, Geuze HJ (2007) Cryosectioning and immunolabeling. Nat Protoc 2:2480–2491
Bos E, Sant´Anna C, Gnaegi H, Pinto RF et al (2011) A new approach to improve the quality of ultrathin cryo-sections; its use for immunogold EM and correlative electron cryo-tomography. J Struct Biol 175:62–67
McLean W, Nakane PF (1974) Periodate–lysine–paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem 22:1077–1083
Wilson SM, Bacic A (2012) Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes. Nat Protoc 7:1716–1727
Liou W, Geuze HJ, Slot JW (1996) Improving structural integrity of cryosections for immuno-gold labeling. Histochem Cell Biol 106:41–58
Ding B, Turgeon R, Parthasarathy MV (1991) Routine cryofixation of plant tissue by propane jet freezing for freeze substitution. J Electron Microsc Tech 19:107–117
Hainachi T, Sato T, Hoshimo M, Mizumo N (1986) A stable lead stain by modification of Sato’s method. Proc. XIth Int. Cong Electron Microsc Kyoto 2181–2182
Griffith JM, Posthuma G (2002) A reliable and convenient method to store ultrathin thawed cryosections prior to immunolabeling. J Histochem Cytochem 50:57–62
Han Y, Yu J, Guo FL, Watkins SC (2006) Polysomes are associated with microtubules in fertilized eggs of Chinese pine (Pinus tabulaeformis). Protoplasma 227:223–227
Guo FL, Yu L, Watkins SC, Han YZ (2007) Orientation of microtubules suggests a role in mRNA transportation in fertilized eggs of Chinese pine (Pinus tabulaeformis). Protoplasma 231:239–243
Yang D, Guo FL, Liu B, Huang H, Watkins SC (2003) Expression and localization of human lysozyme in the endosperm of transgenic rice. Planta 216:597–603
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer International Publishing Switzerland
About this chapter
Cite this chapter
Guo, F., Huang, B. (2015). Immunogold Labeling for Electron Microscopy: Strategy and Problem Solving. In: Yeung, E., Stasolla, C., Sumner, M., Huang, B. (eds) Plant Microtechniques and Protocols. Springer, Cham. https://doi.org/10.1007/978-3-319-19944-3_14
Download citation
DOI: https://doi.org/10.1007/978-3-319-19944-3_14
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-19943-6
Online ISBN: 978-3-319-19944-3
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)