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This protocol allows the multiplexed use of four different molecular labelling techniques in whole-mount Platynereis tissues. In short, gene expression (via in situ HCR 3.0), cell proliferation (via EdU labelling), proteins (via Immunohistochemistry) and nuclei (DAPI/Hoechst staining) can be assessed in whole-mount tissue samples, allowing the co-visualisation of various types of molecules in the same specimen. This protocol combines knowledge from multiple sources (see below), and is being submitted in parallel with a manuscript detailing the applications of the method. (We will add the reference as soon as it's available.) We successfully applied this protocol to heads and posterior regenerates of the bristleworm Platynereis dumerilii, at various developmental stages of the animal. Given the general nature of the detected molecules, and the wide-spread use of the individual detection techniques, we anticipate that this protocol will be well applicable to a wider range of model systems. References: 1) Choi HMT, Calvert CR, Husain N, Huss D, Barsi JC, Deverman BE, Hunter RC, Kato M, Lee SM, Abelin ACT, Rosenthal AZ, Akbari OS, Li Y, Hay BA, Sternberg PW, Patterson PH, Davidson EH, Mazmanian SK, Prober DA, Rijn M van de, Leadbetter JR, Newman DK, Readhead C, Bronner ME, Wold B, Lansford R, Sauka-Spengler T, Fraser SE, Pierce NA. 2016. Mapping a multiplexed zoo of mRNA expression. Development 143:3632–3637. doi:10.1242/dev.140137 2) Choi HMT, Schwarzkopf M, Fornace ME, Acharya A, Artavanis G, Stegmaier J, Cunha A, Pierce NA. 2018. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145:dev165753. doi:10.1242/dev.165753 3) Kuehn E, Clausen DS, Null RW, Metzger BM, Willis AD, Özpolat BD. 2021. Segment number threshold determines juvenile onset of germline cluster proliferation in Platynereis dumerilii. Biorxiv 2021.04.22.439825. doi:10.1101/2021.04.22.439825 4) Pende M, Vadiwala K, Schmidbaur H, Stockinger AW, Murawala P, Saghafi S, Dekens MPS, Becker K, Revilla-i-Domingo R, Papadopoulos S-C, Zurl M, Pasierbek P, Simakov O, Tanaka EM, Raible F, Dodt H-U. 2020. A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity. Sci Adv 6:eaba0365. doi:10.1126/sciadv.aba0365 5) Salic A, Mitchison TJ. 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc National Acad Sci 105:2415–2420. doi:10.1073/pnas.0712168105 6) Tessmar-Raible K, Steinmetz PRH, Snyman H, Hassel M, Arendt D. 2005. Fluorescent two-color whole mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development. Biotechniques 39:460–464. doi:10.2144/000112023 |
