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Updated: Jun 11, 2026
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Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections
Published on: June 25, 2021
A quantitative in situ hybridization protocol for formalin-fixed paraffin-embedded archival post-mortem human brain
Chun-Qing Liu1, Ling Shan, Rawien Balesar
1Department of Neurobiology, Institute of Neuroscience, Zhejiang University School of Medicine, Hangzhou, China.
Methods (San Diego, Calif.)
|July 13, 2010
Summary
This study optimized radioactive in situ hybridization (ISH) for human brain tissue, significantly reducing background noise. The improved method enhances quantification of low-abundance mRNA, like histidine decarboxylase (HDC), for better gene expression analysis.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Radioactive in situ hybridization (ISH) is crucial for quantifying mRNA in archival human brain tissue.
- Non-specific deposits in ISH create background speckles, limiting accurate measurement of low-to-moderate abundant mRNA.
- Accurate gene expression analysis is vital for understanding neurological diseases.
Purpose of the Study:
- To optimize radioactive ISH protocols for enhanced mRNA quantification in formalin-fixed, paraffin-embedded human brain tissue.
- To reduce non-specific background deposits and improve signal-to-noise ratios for low-abundant gene detection.
- To validate the improved protocol for histidine decarboxylase (HDC) mRNA quantification and assess its broader applicability.
Main Methods:
- Modified a standard radioactive ISH protocol by omitting glycogen during probe purification.
- Dissolved precipitated probes in water before adding hybridization buffer (HBF).
- Increased dithiothreitol (DTT) concentration in HBF to 800 mM and elevated hybridization/wash temperatures to enhance stringency.
Main Results:
- The optimized protocol significantly reduced background noise, resulting in clearer ISH images.
- Signal-to-noise ratios were substantially improved (p=0.001) compared to the standard protocol.
- The enhanced protocol successfully quantified HDC mRNA and corticotrophin-releasing hormone mRNA in human brain tissue.
Conclusions:
- The modified radioactive ISH protocol effectively minimizes background noise and enhances signal detection in archival human brain tissue.
- This optimized method provides a more reliable approach for quantifying low-to-moderate abundant mRNA expression.
- The protocol's applicability extends to other low-abundant genes in both brain and potentially other tissue types.
