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

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2023 Jul 10

PMID 37428375

Authors

Markus A Grohme

Olga Frank

Jochen C Rink

Affiliations

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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.

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