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Updated: Jun 3, 2025
Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window
Published on: November 18, 2010
Protocol for the isolation of brain microvessels and visualization of RNA fluorescence in mice and humans
Olivia M Osborne1, Oandy Naranjo1, Silvia Torices1
1University of Miami Miller School of Medicine, Department of Biochemistry and Molecular Biology, Miami, FL, USA.
Abstract:
Here, we present a protocol for isolating microvessels from fresh or snap-frozen brain tissue from mice and humans, followed by visualization of RNA utilizing RNAscope hybridization for quantification of mRNA. We describe the steps for sample preparation and isolation, fixation, and hybridization. This protocol was specifically designed to integrate with RNAscope in situ hybridization. Although the protocol was developed in a mouse model, it can be optimized for use in other organisms, including human brain samples.
Insights
This study details a protocol for isolating brain microvessels and visualizing messenger RNA (mRNA) using RNAscope hybridization. This method allows for accurate mRNA quantification in both mouse and human brain tissue samples.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Microvessels in the brain play crucial roles in its function and are implicated in various neurological diseases.
- Accurate quantification of messenger RNA (mRNA) within these microvessels is essential for understanding gene expression patterns and disease mechanisms.
- Existing methods for microvessel isolation and subsequent RNA analysis can be complex and time-consuming.
Purpose of the Study:
- To present a standardized protocol for the isolation of microvessels from brain tissue.
- To enable the visualization and quantification of mRNA within isolated microvessels using RNAscope in situ hybridization.
- To provide a method applicable to both fresh and snap-frozen brain samples from mice and humans.
Main Methods:
- Isolation of microvessels from fresh or snap-frozen brain tissue (mouse and human).
- Sample preparation, including fixation, for optimal RNA preservation.
- RNAscope hybridization technique for in situ visualization and quantification of mRNA.
Main Results:
- A detailed protocol for microvessel isolation and RNAscope integration is described.
- The protocol facilitates the quantification of mRNA within brain microvessels.
- The method has been validated in mouse models and is adaptable for human brain samples.
Conclusions:
- This protocol offers a robust method for studying gene expression in brain microvessels.
- It facilitates a deeper understanding of microvessel biology in neurological health and disease.
- The adaptability to human samples makes it valuable for translational research.
