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A simple fixation and embedding method for use in hybridization histochemistry on plant tissues
G I McFadden1, I Bonig, E C Cornish
1Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria, Australia.
The Histochemical Journal
|October 1, 1988
Summary
A new hybridization histochemistry method fixes and embeds plant tissues, preserving nucleic acids for RNA localization. This technique enables detailed transcript analysis at the electron microscopic level using labeled DNA probes.
Area of Science:
- Plant Biology
- Molecular Biology
- Histochemistry
Background:
- Hybridization histochemistry is crucial for localizing nucleic acids within biological tissues.
- Existing methods for plant tissue fixation and embedding can compromise nucleic acid integrity.
Purpose of the Study:
- To develop a simple and effective method for fixing and embedding plant tissue for hybridization histochemistry.
- To ensure preservation of nucleic acids in situ for subsequent analysis.
Main Methods:
- Plant tissue was fixed using paraformaldehyde-glutaraldehyde via the phase-partition technique.
- Embedding was performed using LR Gold resin.
- Double-stranded 32P-labeled DNA probes were hybridized to complementary RNAs in thin and semithin sections.
- Biotinylated probes with immunogold markers were used for electron microscopy.
Main Results:
- The developed method successfully preserves nucleic acids in plant tissues.
- Hybridization with labeled DNA probes allowed for the detection of complementary RNAs.
- Electron microscopy revealed the precise localization of transcripts at the ultrastructural level.
Conclusions:
- This simple fixation and embedding method is suitable for hybridization histochemistry in plant tissues.
- The technique effectively preserves nucleic acids, enabling high-resolution transcript localization.
- It offers a valuable tool for studying gene expression in plants at the molecular and ultrastructural levels.