15 Electron Microscopic in Situ Hybridization and its Combination
219
mediately frozen sections. Based on this assessment of maintaining tissue morphology and retaining the messages, we utilize the
preembedding electron microscopic in situ hybridization method using 6 /lm frozen sections fixed in 4% paraformaldehyde for
the simultaneous detection of mRNA and encoded protein.
In general, osmification and embedment in Epon resin are
reported to decrease the immunoreactivity of the targeted protein. However, immunoreactivity can be retrieved by the etching
process using H20 2 or sodium periodate even after modification
such as osmification and embedment in Epon resin, and tissues
embedded in Epon resin can be served for the ultrastructural
simultaneous detection of messages and encoded proteins.
The only problem is the deosmification and degradation of
the signals of mRNA, which are caused by etching process using
H20 2 or sodium periodate. In order to resolve this problem, we
have recently used LR White resin for tissue embedment (Matsuno et al. 1998). In LR White resin-embedded tissues, retrieval
of immunoreactivity using H20 2 or sodium periodate is not required, and therefore the degradation of the signals of mRNA
can be avoided.
To our knowledge, only five reports describing the ultrastructural simultaneous detection of mRNA and encoded protein
have been published, in each of which postembedding electron
microscopic in situ hybridization method using colloidal gold
particle was utilized. Some investigators (Singer et al. 1989;
Escaig-Haye et al. 1992; Egger et al. 1994; Gingras & Bendayan
1995) utilized Lowicryl K4M-embedded tissues for the detection
of messages, while others (Morey et al. 1995) utilized LR Whiteembedded tissues. However, in their postembedding electron
microscopic in situ hybridization studies, the relatively frequent
non-specific reactions of colloidal gold particles used for the detection of mRNA were observed in the cisternae of the rough
endoplasmic reticulum. Electron microscopic in situ hybridization method for Lowicryl K4M-embedded tissues is generally
supposed to present some difficulty in morphological preservation. From the viewpoint of specific hybridization signal detection and morphological preservation, combined immunohistochemistry and pre embedding electron microscopic in situ
hybridization method is supposed to be superior to the previously reported postembedding electron microscopic in situ
hybridization method. This ultrastructural double staining
219
mediately frozen sections. Based on this assessment of maintaining tissue morphology and retaining the messages, we utilize the
preembedding electron microscopic in situ hybridization method using 6 /lm frozen sections fixed in 4% paraformaldehyde for
the simultaneous detection of mRNA and encoded protein.
In general, osmification and embedment in Epon resin are
reported to decrease the immunoreactivity of the targeted protein. However, immunoreactivity can be retrieved by the etching
process using H20 2 or sodium periodate even after modification
such as osmification and embedment in Epon resin, and tissues
embedded in Epon resin can be served for the ultrastructural
simultaneous detection of messages and encoded proteins.
The only problem is the deosmification and degradation of
the signals of mRNA, which are caused by etching process using
H20 2 or sodium periodate. In order to resolve this problem, we
have recently used LR White resin for tissue embedment (Matsuno et al. 1998). In LR White resin-embedded tissues, retrieval
of immunoreactivity using H20 2 or sodium periodate is not required, and therefore the degradation of the signals of mRNA
can be avoided.
To our knowledge, only five reports describing the ultrastructural simultaneous detection of mRNA and encoded protein
have been published, in each of which postembedding electron
microscopic in situ hybridization method using colloidal gold
particle was utilized. Some investigators (Singer et al. 1989;
Escaig-Haye et al. 1992; Egger et al. 1994; Gingras & Bendayan
1995) utilized Lowicryl K4M-embedded tissues for the detection
of messages, while others (Morey et al. 1995) utilized LR Whiteembedded tissues. However, in their postembedding electron
microscopic in situ hybridization studies, the relatively frequent
non-specific reactions of colloidal gold particles used for the detection of mRNA were observed in the cisternae of the rough
endoplasmic reticulum. Electron microscopic in situ hybridization method for Lowicryl K4M-embedded tissues is generally
supposed to present some difficulty in morphological preservation. From the viewpoint of specific hybridization signal detection and morphological preservation, combined immunohistochemistry and pre embedding electron microscopic in situ
hybridization method is supposed to be superior to the previously reported postembedding electron microscopic in situ
hybridization method. This ultrastructural double staining