Immunogold Labeling for Electron Microscopy: Strategy and Problem Solving

  • Fengli GuoEmail author
  • Bing Quan Huang


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.


Cryofixation–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.


  1. 1.
    Griffiths G (1993) Fine structure immunocytochemistry. Springer-Verlag, BerlinCrossRefGoogle Scholar
  2. 2.
    Tokuyasu KT (1973) A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 57:551–565PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Tokuyasu KT (1986) Application of cryoultramicrotomy to immunocytochemistry. J Microsc 143:139–149CrossRefPubMedGoogle Scholar
  4. 4.
    Kiss JZ, McDonald K (1993) Electron microscopy immunocytochemistry flowing cryofixation and freeze substitution. Meth Cell Biol 37:311–341CrossRefGoogle Scholar
  5. 5.
    Newman GR, Hobot JA (1987) Modern acrylics for post-embedding immunostaining techniques. J Histochem Cytochem 35:971–981CrossRefPubMedGoogle Scholar
  6. 6.
    Newman GR, Hobot JA (2001) Resin microscopy and on-section immunocytochemistry. Springer–Verlag, New YorkCrossRefGoogle Scholar
  7. 7.
    McDonald K (1999) High-pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabeling. Meth Mol Biol 117:77–97CrossRefGoogle Scholar
  8. 8.
    McDonald K (2007) Cryopreparation methods for electron microscopy of selected model systems. Meth Cell Biol 79:23–56CrossRefGoogle Scholar
  9. 9.
    McDonald K (2014) Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material. Protoplasma 251:429–448CrossRefPubMedGoogle Scholar
  10. 10.
    Takeuchi M, Takabe K, Momeyuki Y (2010) Immunoelectron microscopy of cryofixed and freeze substituted plant tissues. Meth Mol Biol 657:155–165CrossRefGoogle Scholar
  11. 11.
    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 147–157CrossRefGoogle Scholar
  12. 12.
    Vaughn KC, Campbell WH (1988) Immunogold localization of nitrate reductase in maize leaves. Plant Physiol 88:1354–1357PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Kuo J (2007) Processing tissues for ultrastructural study in electron microscopy: methods and protocols. Humana Press, New YorkGoogle Scholar
  14. 14.
    Hayat MA (2000) Principles and techniques of electron microscopy: biological applications, 4th edn. Cambridge University Press, New YorkGoogle Scholar
  15. 15.
    Ripper D, Schwarzt H, Stierhof YD (2008) Cryo-section immunolabeling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration. Biol Cell 100:109–123CrossRefPubMedGoogle Scholar
  16. 16.
    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 62:423–434CrossRefGoogle Scholar
  17. 17.
    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 18:715–730PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Slot J, Geuze HJ (2007) Cryosectioning and immunolabeling. Nat Protoc 2:2480–2491CrossRefPubMedGoogle Scholar
  19. 19.
    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 175:62–67CrossRefPubMedGoogle Scholar
  20. 20.
    McLean W, Nakane PF (1974) Periodate–lysine–paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem 22:1077–1083CrossRefPubMedGoogle Scholar
  21. 21.
    Wilson SM, Bacic A (2012) Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes. Nat Protoc 7:1716–1727CrossRefPubMedGoogle Scholar
  22. 22.
    Liou W, Geuze HJ, Slot JW (1996) Improving structural integrity of cryosections for immuno-gold labeling. Histochem Cell Biol 106:41–58CrossRefPubMedGoogle Scholar
  23. 23.
    Ding B, Turgeon R, Parthasarathy MV (1991) Routine cryofixation of plant tissue by propane jet freezing for freeze substitution. J Electron Microsc Tech 19:107–117CrossRefPubMedGoogle Scholar
  24. 24.
    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 2181–2182Google Scholar
  25. 25.
    Griffith JM, Posthuma G (2002) A reliable and convenient method to store ultrathin thawed cryosections prior to immunolabeling. J Histochem Cytochem 50:57–62CrossRefPubMedGoogle Scholar
  26. 26.
    Han Y, Yu J, Guo FL, Watkins SC (2006) Polysomes are associated with microtubules in fertilized eggs of Chinese pine (Pinus tabulaeformis). Protoplasma 227:223–227CrossRefPubMedGoogle Scholar
  27. 27.
    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 231:239–243CrossRefPubMedGoogle Scholar
  28. 28.
    Yang D, Guo FL, Liu B, Huang H, Watkins SC (2003) Expression and localization of human lysozyme in the endosperm of transgenic rice. Planta 216:597–603PubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Stowers Institute for Medical ResearchKansas CityUSA
  2. 2.Center for Basic Research in Digestive DiseaseMayo ClinicRochesterUSA

Personalised recommendations