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Updated: Aug 8, 2026
Flash Freezing and Cryosectioning E12.5 Mouse Brain
Published on: May 28, 2007
Just cool it! Cryoprotectant anti-freeze in immunocytochemistry and in situ hybridization
1Department of Anatomy and Neurobiology, School of Medicine, University of Maryland, 685 W Baltimore Street, Baltimore, MD 21201, USA. gehoffma@umaryland.edu
Insights
Storing tissue sections in an anti-freeze cryoprotectant solution at -20°C for years preserves antigenicity. This method enables advanced techniques like non-radioactive in situ hybridization (ISH) for neuroscience research.
Area of Science:
- Neuroscience
- Immunohistochemistry
- Molecular Biology
Background:
- Immunohistochemical techniques are vital for visualizing antigens in tissues, offering high specificity and signal-to-noise ratios.
- Traditional methods faced challenges with antigenicity loss and immediate processing requirements for tissue sections.
- Limited storage options, like sucrose solutions, posed risks of degradation and compromised tissue integrity.
Purpose of the Study:
- To review advances in immunocytochemical technology and tissue storage.
- To highlight the long-term preservation of antigenicity in cryoprotectant solutions.
- To showcase the application of these methods in modern neuroscience research.
Main Methods:
- Development of an anti-freeze cryoprotectant solution for long-term tissue section storage at -20°C.
- Testing of new non-radioactive in situ hybridization (ISH) techniques on long-term stored tissue.
- Application of immunocytochemistry principles to enhance tissue analysis.
Main Results:
- Tissue sections stored for many years in anti-freeze cryoprotectant solutions retain their antigenicity.
- Modern non-radioactive ISH techniques are compatible with long-term stored tissue sections.
- The cryoprotectant method has become a widely adopted gold standard for tissue preservation.
Conclusions:
- Long-term storage of tissue sections in anti-freeze cryoprotectant solutions is a robust method for preserving antigenicity.
- This technique significantly enhances the utility of stored tissues for advanced molecular analyses, including ISH.
- Advances in immunocytochemistry and tissue preservation have revolutionized neuroscience applications.
Abstract:
Immunohistochemical techniques offer specificity as well as flexibility for visualizing antigens. Their use with freely floating sections provides a high signal-to-noise ratio and has become a gold standard for brain and a number of other tissues. Yet this approach initially suffered from inability to keep the antigenicity in tissue sections and required immediate processing of all cut sections. Use of sucrose solutions enabled storage at refrigerator temperatures for a few days but longer-term storage was risky and either bacterial/fungal growth or evaporation of the storage solution compromised the integrity of the tissue. Our discovery 25 years ago that tissue sections can be stored for many years at -20 degrees C in an anti-freeze cryoprotectant solution with no loss of antigenicity solved this problem and has become widely used. More recently the utility of tissue stored for many years in anti-freeze cryoprotectant was pushed to new levels by testing new non-radioactive in situ hybridization (ISH) techniques that are based on modern immunocytochemistry. This review touches upon these advances in immunocytochemical technology using examples from neuroscience applications.
