Preprint Technical Note Version 1 Preserved in Portico This version is not peer-reviewed

Preparing Planarian Cells for High-content Fluorescence Microscopy Using RNA in situ Hybridization and Immunocytochemistry

Version 1 : Received: 10 November 2021 / Approved: 15 November 2021 / Online: 15 November 2021 (11:44:53 CET)

How to cite: Grohme, M.; Frank, O.; Rink, J. Preparing Planarian Cells for High-content Fluorescence Microscopy Using RNA in situ Hybridization and Immunocytochemistry. Preprints 2021, 2021110256 (doi: 10.20944/preprints202111.0256.v1). Grohme, M.; Frank, O.; Rink, J. Preparing Planarian Cells for High-content Fluorescence Microscopy Using RNA in situ Hybridization and Immunocytochemistry. Preprints 2021, 2021110256 (doi: 10.20944/preprints202111.0256.v1).

Abstract

High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. The planarian community has developed sensitive and robust assays for whole animals, yet cell based assays, despite their practical aspects, have not been explored to the same extent. Here we describe a modular collection of detailed protocols adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. Methods include the detection of RNA transcripts by RNA fluorescent in situ hybridization combined with tyramide signal amplification using hapten-labeled riboprobes. In addition, immunocytochemical protocols for quantifying proliferating cells by the detection of phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine incorporation into the nuclear genome are described. 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. Recommendations for successful experimental workflows and common sources of errors are discussed.

Keywords

planarian; maceration; high-content fluorescence microscopy; formaldehyde fixation; RNA FISH; immunocytochemistry; BrdU; phospho-histone H3; tyramide signal amplification

Subject

BIOLOGY, Other

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