Ultrastructural EIH by pre-embedding staining

Almost all pre-embedding staining procedures consist in the following steps (i) prefixation to obtain maximum permeabilization of the tissue with the best acceptable structural preservation and minimal loss of antigens (ii) immunohistochemical incubations (iii) further fixation (iv) revelation of the enzyme (v) postfixation (e.g., with osmic acid to render the DAB product electron dense) and, (vi) dehydration and embedding. Extensive washings are included between the various steps. The enzyme-generated product should be insoluble, both in water and alcohol. 

In the method developed by Pickel et al. (1976), organs or animals are perfused with fixative, followed by the preparation of sections (about 20 pm) which are then treated with detergent (Triton X-100) to increase permeability. Subseqent EIH staining, as in light microscopy (Section 17.3.3), gives suitable results for the outer 2-3 pm layer of thick sections. 

Instead of further increasing permeabilization of the tissues, it is possible to decrease the size of the tracer, by conjugating Fab with MPOase (Section 10.4.1). 

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