15 Electron Microscopic in Situ Hybridization and its Combination
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serve the visualization of intracellular spatial relationship between pituitary hormone and its message at the ultrastructural
level. In this chapter, both preembedding and postembedding
methods of non-radioisotopic electron microscopic in situ hybridization are introduced together with combined immunohistochemistry and preembedding non-radioisotopic in situ hybridization technique.
Outline
The protocol of each of the following techniques is outlined in
Fig. 1-3.
Pre embedding non-radioisotopic electron microscopic m
situ hybridization (Fig. 1)
Postembedding non-radioisotopic electron microscopic m
situ hybridization (Fig. 2)
Combined immunohistochemistry and preembedding nonradioisotopic electron microscopic in situ hybridization (Fig. 3)
Frozen section fixed with 4% PFA
•
Hybridization with biotinylated probe at 37°C overnight
•
Signal detection by ABC-HRP-DAB
•
Osmification
•
Embedment in Epon resin
•
Electron microscopic observation
Fig. 1. Outline of preembedding electron microscopic in situ hybridization.
PFA: paraformaldehyde, ABC-HRP-DAB: streptavidin-biotin complex, horseradish peroxidase and diaminobenzidine