Introduction to Immunohistochemistry




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Electron microscopic (EM) immunohistichemical techniques can be divided into two groups: Those where the immunostaining takes place prior to resin embedding are referred to as pre-embedding. Those methods where the immunolabeling is undertaken after resin embedding are known as post-embedding.


The choice of whether to apply pre- or post-embedding method to the detection of an antigen in any particular location will depend to a large extent upon the distribution and liability of the antigen and the characteristics of the primary antibody. Before starting immuno-EM labeling, a test for the characteristics and dilution of the primary antibody should be performed at light microcopy level. 


Several recently developed methods rely on labeling with colloidal gold particles. These methods were originally introduced for electron microscopy (Faulk and Taylor 1971) as the gold particles are easily visible under the electron microscope, but they are also useful for light microscopy. 


Since gold particles can be made in different size from 5 to 30 nm, it is possible to carry out multiple staining at the electron microscopic level, most easily by direct labeling of several first layer antibodies with different sized particles. The indirect techniques can also be used in double or triple labeling by parallel approach if the primary antibodies are from different species, and by sequential approach if the primary antibodies are from same species.