Preparing Planarian Cells for High-Content Fluorescence Microscopy Using RNA in 
Situ Hybridization and Immunocytochemistry.

Grohme, Markus; Frank, Olga; Rink, Jochen

doi:10.1007/978-1-0716-3275-8_8

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Preparing Planarian Cells for High-Content Fluorescence Microscopy Using RNA in Situ Hybridization and Immunocytochemistry.

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Grohme, Markus
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

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Frank, Olga
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

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Rink, Jochen
Max Planck Institute for Molecular Cell Biology and Genetics, Max Planck Society;

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Citation

Grohme, M., Frank, O., & Rink, J. (2023). Preparing Planarian Cells for High-Content Fluorescence Microscopy Using RNA in Situ Hybridization and Immunocytochemistry. Methods in molecular biology (Clifton, N.J.), 2680, 121-155. doi:10.1007/978-1-0716-3275-8_8.

Cite as: https://hdl.handle.net/21.11116/0000-000E-AB36-5

Abstract

High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. Here we describe a modular collection of assays adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. These include protocols for RNA fluorescent in situ hybridization (RNA FISH) as well as immunocytochemical protocols for quantifying proliferating cells targeting phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine (BrdU) incorporated into the nuclear DNA. The assays are compatible with planarians of virtually any size, as the tissue is disaggregated into a single-cell suspension before fixation and staining. By sharing many reagents with established planarian whole-mount staining protocols, preparation of samples for high-content microscopy adoption requires little additional investment.