Pre-embedding Immunogold Localization of Antigens in Mammalian Brain Slices

  • Thomas Schikorski
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 657)

Abstract

The detection of proteins with antibodies that are conjugated to gold particles has been a major asset to cell biology and the neurosciences, and knowledge about the subcellular location of antigens has formed the basis for many hypotheses regarding protein function. Many protocols have been developed since the introduction of colloidal gold to immunocytochemistry. The two most widely used techniques, however, are based on transmission electron microscopy and consist of either immunolabeling before the specimens are embedded in resin (pre-embedding immunogold labeling) or immunolabeling after embedding in resin (post-embedding immunogold labeling). The following protocol describes a pre-embedding procedure that gives reliable results with all antibodies that produce adequate staining as observed with a light microscope. This procedure results in almost perfect preservation of the ultrastructure. The procedure employs thick sectioning using a vibratome, permeabilization of membranes with Triton X-100, and immunolabeling with fluorescently conjugated Nanogold antibodies, followed by gold enhancement and embedding for electron microscopy. We also discuss some limitations inherent to pre-embedding immunogold labeling.

Key words

Immunohistochemistry electron microscopy fluoro-nanogold gold enhancement synaptic vesicle protein permeabilization correlated light and electron microscopy 

Notes

Acknowledgments

This work was supported by the NIH grant U54 NS039408 and the NIH grant R21 NS0263208.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Thomas Schikorski
    • 1
  1. 1.Neuroscience DepartmentUniversidad Central del CaribeBayamonUSA

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