Publication: Electron microscopic observation of intracellular expression of mRNA and its protein product: Technical
review on ultrastructural in situ hybridization and its
combination with immunohistochemistry
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Date
2000
Authors
Matsuno, A. ; Nagashima, T. ; Ohsugi, Y. ; Utsunomiya, H. ; Takekoshi, S. ; Munakata, S. ; Nagao, K. ; Osamura, R.Y. ; Watanabe, K.
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Publisher
Murcia : F. Hernández
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DOI
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info:eu-repo/semantics/article
Description
Abstract
In situ hybridization (ISH) at the electron
microscopic (EM) level is essential for elucidating the
intracellular distribution and role of mRNA in protein
synthesis. Three different approaches have been applied
by the investigators in this EM-ISH study: preembedding
method; non-embedding method using
ultrathin frozen sections; and postembedding method. In
order to obtain satisfactory morphological preservation
and retain the messages, we routinely utilized 6 pmthick
frozen sections fixed in 4% paraformaldehyde for
the preembedding method and tissues embedded in LR
White resin for the postembedding method. The
hybridization signal intensity by the postembedding
method was lower, and non-specific signals were
relatively frequent, in comparison with the preembedding
method. The preembedding method thus
appears to be easier and better than the postembedding
method from the viewpoint of applicability and
preservation of mRNA, although quantitative analysis of
the expression of mRNA is rather difficult in the
preembedding method. EM-ISH is considered to be an
important tool for clarifying the intracellular localization
of mRNA and the exact site of specific hormone
synthesis on the rough endoplasmic reticulum. The
simultaneous visualization of mRNA and encoded
protein in the same cells using preembedding EM-ISH and subsequent postembedding immunoreaction with
protein A colloidal gold complex is also described. This
ultrastructural double-staining method for mRNA and
encoded protein can be expected to provide an important clue for elucidating the intracellular correlation of
mRNA translation and secretion of translated protein.
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