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In situ hybridization histochemistry quantification: automatic count on single cell in digital image
M Masseroli1, A Bollea, C Bendotti
1Department of Geriatric Neuropsychiatry, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
Journal of Neuroscience Methods
|April 1, 1993
Summary
This study introduces a new method for quantifying mRNA expression in cells using in situ hybridization and digital image analysis. The technique accurately measures radioactive hybrids, enhancing sensitivity for detecting subtle changes in neuronal function.
Area of Science:
- Neuroscience
- Molecular Biology
- Biotechnology
Background:
- In situ hybridization is vital for studying central nervous system (CNS) mRNA expression.
- Quantitative evaluation of this technique is challenging due to difficulties in precise measurement.
Purpose of the Study:
- To develop and validate a reliable method for quantifying radioactive hybrids in individual cells using digital image analysis.
- To improve the sensitivity and quantitative accuracy of in situ hybridization for neuroscience research.
Main Methods:
- A novel method quantifying radioactive hybrids via silver grain count in digital images.
- Utilizing computerized microscope image analysis (IBAS 2) for automatic cell area, grain portion, and grey level identification.
- Implementing a mathematical function with a 'density factor' to accurately count overlapping grains.
Main Results:
- The method successfully quantified preproNPY mRNA in rat dentate gyrus and preproSomatostatin mRNA in the frontal cortex.
- Colchicine treatment reduced per-cell expression of preproSomatostatin mRNA without altering positive cell numbers.
- The technique demonstrated accurate quantification across a wide range of silver grains (10-5000).
Conclusions:
- The developed method offers a significant improvement in the quantitative capabilities of in situ hybridization.
- This approach enhances sensitivity, enabling the detection of small alterations in neuronal function.
- The technique establishes in situ hybridization as a robust quantitative tool for CNS research.