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Planarian Regeneration pp 353–366Cite as

Fixation, Processing, and Immunofluorescent Labeling of Whole Mount Planarians

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1774))

Abstract

Efforts to elucidate mechanisms of regeneration in the planarian Schmidtea mediterranea have included the application of immunocytochemical methods to detect specific molecules and label cells and tissues in situ. Here we describe methods for immunofluorescent labeling of whole mount planarians. We outline protocols for fixation and steps for processing animals prior to immunolabeling, incorporating commonly utilized reagents for mucus removal, pigment bleaching, tissue permeabilization, and antigen retrieval. Because processing steps can mask or degrade antigens, we also recommend protocol parameters that can be tested simultaneously to optimize sample preparation for novel antibodies.

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References

  1. Guo T, Peters AH, Newmark PA (2006) A Bruno-like gene is required for stem cell maintenance in planarians. Dev Cell 11(2):159–169. https://doi.org/10.1016/j.devcel.2006.06.004

    Article  CAS  PubMed  Google Scholar 

  2. Orii H, Ito H, Watanabe K (2002) Anatomy of the planarian Dugesia japonica I. The muscular system revealed by antisera against myosin heavy chains. Zool Sci 19(10):1123–1131. https://doi.org/10.2108/zsj.19.1123

    Article  CAS  PubMed  Google Scholar 

  3. Orii H, Sakurai T, Watanabe K (2005) Distribution of the stem cells (neoblasts) in the planarian Dugesia japonica. Dev Genes Evol 215(3):143–157. https://doi.org/10.1007/s00427-004-0460-y

    Article  CAS  PubMed  Google Scholar 

  4. Nishimura K, Yamamoto H, Kitamura Y, Agata K (2008) Brain and neural networks. In: Raffa RB, Rawls SM (eds) Planaria: a model for drug action and abuse. Landes Bioscience, Austin, pp 4–12

    Google Scholar 

  5. Ito H, Saito Y, Watanabe K, Orii H (2001) Epimorphic regeneration of the distal part of the planarian pharynx. Dev Genes Evol 211(1):2–9. https://doi.org/10.1007/s004270000115

    Article  CAS  PubMed  Google Scholar 

  6. Cebrià F (2008) Organization of the nervous system in the model planarian Schmidtea mediterranea: an immunocytochemical study. Neurosci Res 61(4):375–384. https://doi.org/10.1016/j.neures.2008.04.005

    Article  CAS  PubMed  Google Scholar 

  7. Robb SM, Sánchez Alvarado A (2002) Identification of immunological reagents for use in the study of freshwater planarians by means of whole-mount immunofluorescence and confocal microscopy. Genesis 32(4):293–298. https://doi.org/10.1002/gene.10087

  8. Fraguas S, Barberán S, Ibarra B, Stöger L, Cebrià F (2012) Regeneration of neuronal cell types in Schmidtea mediterranea: an immunohistochemical and expression study. Int J Dev Biol 56(1–3):143–153. https://doi.org/10.1387/ijdb.113428sf

    Article  CAS  PubMed  Google Scholar 

  9. Newmark PA, Sánchez Alvarado A (2000) Bromodeoxyuridine specifically labels the regenerative stem cells of planarians. Dev Biol 220(2):142–153. https://doi.org/10.1006/dbio.2000.9645

    Article  CAS  PubMed  Google Scholar 

  10. Zayas RM, Cebrià F, Guo T, Feng J, Newmark PA (2010) The use of lectins as markers for differentiated secretory cells in planarians. Dev Dyn 239(11):2888–2897. https://doi.org/10.1002/dvdy.22427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bueno D, Baguñà J, Romero R (1997) Cell-, tissue-, and position-specific monoclonal antibodies against the planarian Dugesia (Girardia) tigrina. Histochem Cell Biol 107(2):139–149. https://doi.org/10.1007/s004180050098

    Article  CAS  PubMed  Google Scholar 

  12. Moritz S, Stockle F, Ortmeier C, Schmitz H, Rodriguez-Esteban G, Key G, Gentile L (2012) Heterogeneity of planarian stem cells in the S/G2/M phase. Int J Dev Biol 56(1–3):117–125. https://doi.org/10.1387/ijdb.113440sm

    Article  CAS  PubMed  Google Scholar 

  13. Romero R, Fibla J, Bueno D, Sumoy L, Soriano MA, Baguna J (1991) Monoclonal-antibodies as markers of specific cell-types and regional antigens in the fresh-water planarian Dugesia (G) Tigrina. Hydrobiologia 227:73–79. https://doi.org/10.1007/Bf00027585

    Article  Google Scholar 

  14. Umesono Y, Watanabe K, Agata K (1997) A planarian orthopedia homolog is specifically expressed in the branch region of both the mature and regenerating brain. Develop Growth Differ 39(6):723–727. https://doi.org/10.1046/j.1440-169X.1997.t01-5-00008.x

    Article  CAS  PubMed  Google Scholar 

  15. Pearson BJ, Eisenhoffer GT, Gurley KA, Rink JC, Miller DE, Sánchez Alvarado A (2009) Formaldehyde-based whole-mount in situ hybridization method for planarians. Dev Dyn 238(2):443–450. https://doi.org/10.1002/dvdy.21849

    Article  PubMed  PubMed Central  Google Scholar 

  16. Tazaki A, Kato K, Orii H, Agata K, Watanabe K (2002) The body margin of the planarian Dugesia japonica: characterization by the expression of an intermediate filament gene. Dev Genes Evol 212(8):365–373. https://doi.org/10.1007/s00427-002-0253-0

    Article  CAS  PubMed  Google Scholar 

  17. Pellettieri J, Fitzgerald P, Watanabe S, Mancuso J, Green DR, Sánchez Alvarado A (2010) Cell death and tissue remodeling in planarian regeneration. Dev Biol 338(1):76–85. https://doi.org/10.1016/j.ydbio.2009.09.015

    Article  CAS  PubMed  Google Scholar 

  18. Cebrià F, Newmark PA (2005) Planarian homologs of netrin and netrin receptor are required for proper regeneration of the central nervous system and the maintenance of nervous system architecture. Development 132(16):3691–3703. https://doi.org/10.1242/dev.01941

    Article  CAS  PubMed  Google Scholar 

  19. Sánchez Alvarado A, Newmark PA (1999) Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc Natl Acad Sci U S A 96(9):5049–5054. https://doi.org/10.1073/pnas.96.9.5049

    Article  CAS  Google Scholar 

  20. Forsthoefel DJ, James NP, Escobar DJ, Stary JM, Vieira AP, Waters FA, Newmark PA (2012) An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians. Dev Cell 23(4):691–704. https://doi.org/10.1016/j.devcel.2012.09.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Willier BH, Hyman LH, Rifenburgh SA (1925) A histochemical study of intracellular digestion in triclad flatworms. J Morphol Physiol 40(2):299–340. https://doi.org/10.1002/Jmor.1050400205

    Article  Google Scholar 

  22. Kobayashi C, Kobayashi S, Orii H, Watanabe K, Agata K (1998) Identification of two distinct muscles in the planarian Dugesia japonica by their expression of myosin heavy chain genes. Zoological Science 15(6):861–869. https://doi.org/10.2108/Zsj.15.861

    Article  CAS  Google Scholar 

  23. Bueno D, Espinosa L, Baguñà J, Romero R (1997) Planarian pharynx regeneration in regenerating tail fragments monitored with cell-specific monoclonal antibodies. Dev Genes Evol 206:425–434. https://doi.org/10.1007/s004270050072

    Article  CAS  PubMed  Google Scholar 

  24. Forsthoefel DJ, Waters FA, Newmark PA (2014) Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling. BMC Dev Biol 14:45. https://doi.org/10.1186/s12861-014-0045-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ross KG, Omuro KC, Taylor MR, Munday RK, Hubert A, King RS, Zayas RM (2015) Novel monoclonal antibodies to study tissue regeneration in planarians. BMC Dev Biol 15:2. https://doi.org/10.1186/s12861-014-0050-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ramos-Vara JA, Beissenherz ME (2000) Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers. J Vet Diagn Investig 12(4):307–311. https://doi.org/10.1177/104063870001200402

    Article  CAS  PubMed  Google Scholar 

  27. Stadler C, Skogs M, Brismar H, Uhlen M, Lundberg E (2010) A single fixation protocol for proteome-wide immunofluorescence localization studies. J Proteome 73(6):1067–1078. https://doi.org/10.1016/j.jprot.2009.10.012

    Article  CAS  Google Scholar 

  28. Duerr JS (2006) Immunohistochemistry (June 19, 2006). In: The C. elegans Research Community (ed) WormBook. WormBook, Pasadena. https://doi.org/10.1895/wormbook.1.105.1. http://www.wormbook.org

    Chapter  Google Scholar 

  29. Hopman AH, Ramaekers FC, Speel EJ (1998) Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for In situ hybridization using CARD amplification. J Histochem Cytochem 46(6):771–777

    Article  CAS  PubMed  Google Scholar 

  30. King RS, Newmark PA (2013) In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea. BMC Dev Biol 13:8. https://doi.org/10.1186/1471-213X-13-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang Y, Stary JM, Wilhelm JE, Newmark PA (2010) A functional genomic screen in planarians identifies novel regulators of germ cell development. Genes Dev 24(18):2081–2092. https://doi.org/10.1101/gad.1951010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Liu SY, Selck C, Friedrich B, Lutz R, Vila-Farre M, Dahl A, Brandl H, Lakshmanaperumal N, Henry I, Rink JC (2013) Reactivating head regrowth in a regeneration-deficient planarian species. Nature 500(7460):81–84. https://doi.org/10.1038/nature12414

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Roma Munday, Matthew Taylor, and Amy Hubert for testing parameters to optimize whole mount immunostaining in the Zayas Laboratory; Carlo Quintanilla for helping to process the anti-synapsin samples presented here; Forrest Waters for optimization efforts in the Newmark Laboratory; our colleagues in the planarian research community whose contributions led to protocol improvements discussed in this chapter. DJF was supported by National Institutes of Health Ruth L. Kirschstein National Research Service Award F32-DK077469. Work in the Zayas Laboratory was supported by California Institute for Regenerative Medicine Grant RN2-00940-1 to RMZ. PAN is an investigator of the Howard Hughes Medical Institute.

Author information

Author notes

  1. David J. Forsthoefel and Kelly G. Ross contributed equally to this work.

Authors and Affiliations

  1. Functional and Chemical Genomics Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK, USA

    David J. Forsthoefel

  2. Department of Biology, San Diego State University, San Diego, CA, USA

    Kelly G. Ross & Ricardo M. Zayas

  3. Department of Integrative Biology, Howard Hughes Medical Institute, Morgridge Institute for Research, University of Wisconsin–Madison, Madison, WI, USA

    Phillip A. Newmark

Authors
  1. David J. Forsthoefel
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  2. Kelly G. Ross
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  3. Phillip A. Newmark
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  4. Ricardo M. Zayas
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Corresponding authors

Correspondence to David J. Forsthoefel or Ricardo M. Zayas .

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Editors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Jochen C. Rink

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Forsthoefel, D.J., Ross, K.G., Newmark, P.A., Zayas, R.M. (2018). Fixation, Processing, and Immunofluorescent Labeling of Whole Mount Planarians. In: Rink, J. (eds) Planarian Regeneration. Methods in Molecular Biology, vol 1774. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7802-1_10

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  • DOI: https://doi.org/10.1007/978-1-4939-7802-1_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7800-7

  • Online ISBN: 978-1-4939-7802-1

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