Protocol - Pre-embedding Electron Microscopic Immunocytochemistry

Electron microscopic immunocytochemistry takes advantage of the increased resolution of the electron microscope to localize label to specific cellular organelles. Optical microscopes limit resolution to the wavelength of light, about 0.2 p,m or 200 nm. The electron microscope has theoretical resolution of less than 0.2 nm. Practically for biological samples, resolution is limited to about 2 nm because of the 

Immunocytochemistry, DOI 10.1007/978-l-4419-1304-3 15, Springer Science-rBusiness Media, EEC 2010 

To be seen in electron microscopic immunocytochemistry labels must be electron dense. That is, the label must not allow electrons to pass through the label molecule, which thus limits the electron microscopic labels to heavy metals. The most common labels for electron microscopic immunocytochemistry are gold and silver. 

It is possible to use HRP as a label for electron microscopic immunocytochemistry because the DAB-developed chromogen generates a reaction product that is easily stained with heavy metals. While HRP can be used for electron microscopic immunocytochemistry, the DAB reaction product does not show high-resolution distribution of the antigen labeled by the 1° antibody. The HRP method has been used to fill specific neurons and trace their dendrites to determine types of synaptic contacts. However, the diffused nature of the reaction product does not allow it to be associated with specific cellular organelles. Therefore, using HRP for electron microscopic immunocytochemistry will not be discussed here. 

There are two different ways to perform electron microscopic immunocytochemistry pre-embedding and postembedding (Stirling, 1990). Pre-embedding electron microscopic immunocytochemistry applies the antibodies and label to samples just after fixation but before embedding in epoxy resin and sectioning. Postembedding electron microscopic immunocytochemistry applies antibodies and label to thin sections made after the samples have been embedded in epoxy resin and sectioned. 

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