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. Preprints2021, 2021110256. https://doi.org/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. https://doi.org/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. Preprints2021, 2021110256. https://doi.org/10.20944/preprints202111.0256.v1
APA Style
Grohme, M., Frank, O., & Rink, J. (2021). Preparing Planarian Cells for High-content Fluorescence Microscopy Using RNA <em>in situ</em> Hybridization and Immunocytochemistry. Preprints. https://doi.org/10.20944/preprints202111.0256.v1
Chicago/Turabian Style
Grohme, M., Olga Frank and Jochen Rink. 2021 "Preparing Planarian Cells for High-content Fluorescence Microscopy Using RNA <em>in situ</em> Hybridization and Immunocytochemistry" Preprints. https://doi.org/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.
Biology and Life Sciences, Immunology and Microbiology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
