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Preparing Planarian Cells for High-Content Fluorescence Microscopy Using RNA in Situ Hybridization and
Markus A Grohme1, Olga Frank1, Jochen C Rink2
1Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|July 10, 2023
Summary
This study presents new high-content fluorescence microscopy assays for planarian cells. These multiplexed assays quantify biomarkers like proliferating cells using RNA FISH and immunocytochemistry, enabling detailed biological insights.
Area of Science:
- Cell Biology
- Molecular Biology
- Regenerative Biology
Background:
- High-content fluorescence microscopy offers quantitative insights into biological systems.
- Planarian research requires efficient methods for analyzing cellular biomarkers.
- Existing whole-mount staining protocols provide a foundation for new assay development.
Purpose of the Study:
- To develop and validate a modular set of assays for fixed planarian cells.
- To enable multiplexed measurement of multiple biomarkers using high-content microscopy.
- To provide protocols for RNA fluorescent in situ hybridization (RNA FISH) and immunocytochemistry.
Main Methods:
- Adaptation of assays for fixed planarian cells in microwell plates.
- Disaggregation of planarian tissue into single-cell suspensions.
- Implementation of RNA FISH and immunocytochemistry for phosphorylated histone H3 and 5-bromo-2'-deoxyuridine (BrdU).
Main Results:
- Successful development of multiplexed biomarker quantification assays for planarian cells.
- Assays are compatible with planarians of various sizes.
- Established protocols require minimal additional investment by leveraging existing reagents.
Conclusions:
- The developed assays provide a powerful tool for high-content analysis of planarian cells.
- This approach facilitates quantitative measurements of proliferation and gene expression.
- The methods are accessible and scalable for diverse planarian research applications.
