Postembedding electron microscopic immunocytochemistry

Merighi, A. (1992) Postembedding electron microscopic immunocytochemistry, in Electron Microscopic Immunocytochemistry (Polak, J. M. and Priestley, J. V., eds.), Oxford University Press, Oxford, pp. 51-88. 

Ottersen OP (1987) Postembedding light- and electron-microscopic immunocytochemistry of amino acids description of a new model system allowing identical conditions for specificity testing and tissue processing. Exp Brain Rc.v 69 167-174. 

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. 

Understanding the difference between processing for pre-embedding and postembedding electron microscopic immunocytochemistry provides a basis for selecting the better technique (Table 15.1). 

Table 15.1 Conditions for pre-embedding and postembedding electron microscopic immunocytochemistry...

Postembedding electron microscopic immunocytochemistry uses tissue or cultured cells that are chemically fixed, sectioned on a diamond knife, and processed for immunocytochemistry. Here, all procedures and reagents, prior to... 

A second approach to postembedding electron microscopic immunocytochemistry is the use of special aqueous resins, while these resins are still hydrophobic, they tolerate some aqueous material in the tissue. Examples of these resins are LR White or Lowicryl K4M. Another approach with aqueous embedding is to freeze tissue in buffer very rapidly, and to section with a cryo-ultramicrotome. Cryo-ultramicrotomy is a technique requiring considerable training and cannot be used by the novice. 

A major limitation of the postembedding electron microscopic immunocytochemistry is the lack of penetration of the antibodies and label into the section. For densely concentrated antigens, this is not a limitation. For example insulin in secretory vesicles will have sufficient insulin antigen exposed on the surface of the section to label the vesicle (Fig. 15.4b arrows). However, infrequent cytoplasmic proteins may not have enough antigens exposed on the surface that will be seen over background labeling. 

An additional limitation of postembedding electron microscopic immunocytochemistry is its lack of traditional staining for membranes and cellular organelles. Because osmium tetroxide is not used before embedding, membranes can only be seen by negative staining. The use of weaker fixation without high concentration of... 

Postembedding electron microscopic immunocytochemistry - a method that uses antibodies applied to sections after embedding in epoxy resin and sectioning with an ultramicrotome. 

---