Adaptation of a non-radioactive in situ hybridization method to electron microscopy
Standard
Adaptation of a non-radioactive in situ hybridization method to electron microscopy : detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies. / Dörries, U; Bartsch, U; Nolte, C; Roth, J; Schachner, M.
in: Histochemistry, Jahrgang 99, Nr. 3, 03.1993, S. 251-62.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
Harvard
APA
Vancouver
Bibtex
}
RIS
TY - JOUR
T1 - Adaptation of a non-radioactive in situ hybridization method to electron microscopy
T2 - detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies
AU - Dörries, U
AU - Bartsch, U
AU - Nolte, C
AU - Roth, J
AU - Schachner, M
PY - 1993/3
Y1 - 1993/3
N2 - In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a 35S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascin-specific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.
AB - In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a 35S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascin-specific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.
KW - Acrylic Resins
KW - Animals
KW - Astrocytes
KW - Cell Adhesion Molecules, Neuronal
KW - Cerebellum
KW - Digoxigenin
KW - Extracellular Matrix Proteins
KW - Immunohistochemistry
KW - In Situ Hybridization
KW - Mice
KW - Mice, Inbred ICR
KW - Microscopy, Electron
KW - Microtomy
KW - Purkinje Cells
KW - RNA Probes
KW - RNA, Messenger
KW - Sensitivity and Specificity
KW - Sulfur Radioisotopes
KW - Tenascin
KW - Tissue Embedding
KW - Transcription, Genetic
KW - Comparative Study
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
M3 - SCORING: Journal article
C2 - 7684036
VL - 99
SP - 251
EP - 262
IS - 3
ER -