An improved ultrastructural double-staining method for rat growth hormone and its mRNA using LR White resin: a technical note
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A Matsuno1, Y Ohsugi, H Utsunomiya
1Department of Neurosurgery, Teikyo University Ichihara Hospital, Ichihara City, Chiba, Japan.
Abstract:
An improved new method for the simultaneous visualization of mRNA and encoded protein in LR White resin-embedded specimens is described. This pre-embedding electron microscopical in situ hybridization (procedure) localized rat growth hormone mRNA specifically as high electron-density products on the polysomes of the rough endoplasmic reticulum. A subsequent post-embedding immunoreaction, using protein A colloidal gold particles, identified growth hormone as gold particles both in the cisternae of the rough endoplasmic reticulum and on the secretory granules. In our previous report, we used Epon resin for tissue embedment, which required an etching process using hydrogen peroxide or sodium periodate for immunoreactivity retrieval. In general, osmification and embedment in Epon resin are reported to decrease the immunoreactivity of the targeted protein, and the etching process using hydrogen peroxide or sodium periodate results in deosmification and shades off the signals of mRNA. To resolve these problems, we have recently used LR White resin for tissue embedment. In LR White resin-embedded tissues, retrieval of immunoreactivity using hydrogen peroxide or sodium periodate is not required, and, therefore, the gradation of the signals of mRNA can be avoided.
Insights
This study introduces a new method for visualizing mRNA and protein in LR White resin, improving upon older Epon resin techniques. The new approach enhances signal clarity for both mRNA and protein detection in electron microscopy.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Simultaneous visualization of mRNA and encoded protein is crucial for understanding gene expression and protein localization.
- Previous methods using Epon resin for electron microscopy embedding faced challenges with reduced immunoreactivity and signal degradation during mRNA detection.
- The etching process required for Epon resin compromised both mRNA signals and protein immunoreactivity.
Purpose of the Study:
- To develop an improved method for simultaneous visualization of mRNA and its encoded protein in LR White resin-embedded specimens.
- To overcome the limitations of previous Epon resin embedding methods, specifically signal degradation and reduced immunoreactivity.
- To achieve clearer and more reliable detection of both mRNA and protein localization using electron microscopy.
Main Methods:
- Developed a pre-embedding electron microscopical in situ hybridization procedure to localize rat growth hormone mRNA.
- Utilized LR White resin for tissue embedment, eliminating the need for harsh etching steps.
- Performed a subsequent post-embedding immunoreaction with protein A colloidal gold particles to identify growth hormone.
Main Results:
- Rat growth hormone mRNA was specifically localized to polysomes on the rough endoplasmic reticulum.
- Growth hormone protein was identified on gold particles within the rough endoplasmic reticulum cisternae and secretory granules.
- The LR White resin method avoided the deosmification and signal degradation associated with Epon resin etching.
Conclusions:
- LR White resin embedding provides a superior method for simultaneous mRNA and protein visualization via electron microscopy.
- This improved technique preserves signal integrity, allowing for clearer localization of both nucleic acids and proteins.
- The new method offers enhanced reliability for studying gene expression and protein synthesis pathways.
