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Fixation, Processing, and Immunofluorescent Labeling of Whole Mount Planarians

Part of the Methods in Molecular Biology book series (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.

Key words

  • Antibodies
  • Flatworms
  • Immunostaining
  • Histology

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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.

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

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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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