Development Genes and Evolution

, Volume 219, Issue 5, pp 273–279 | Cite as

Use of freeze-cracking in ontogenetic research in Macrostomum lignano (Macrostomida, Rhabditophora)

  • Maxime WillemsEmail author
  • Mieke Boone
  • Marjolein Couvreur
  • Katrien De Mulder
  • Jelka Van Ranst
  • Tom Artois
  • Gaëtan Borgonie
Technical Note


A method for studying whole mount flatworm embryos based on freeze-cracking of the eggs is described. This method allows successful immunohistological and immunocytological studies of whole mount embryos. It does not require the use of sharpened needles or a microinjection system to puncture the eggshell. Moreover, this method is more practical and less time-consuming than classical puncturing and much cheaper than the use of a microinjection system. The advantages of this method are illustrated by results of several immunolocalisation experiments in the macrostomid flatworm Macrostomum lignano. The optimal procedure and crucial steps for this method are discussed.


Platyhelminthes Freeze-cracking Embryonic development Eggshell Immunohistochemistry 



We are very grateful to Dr. Peter Ladurner (University of Innsbruck, Austria) for kindly providing the monoclonal antibodies. M.W. acknowledges the advice of Dr. Bernhard Egger and Dr. Volker Hartenstein regarding the stainings. This work was supported by IWT doctoral grants to M.W. and S.M. by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) and a FWO grant to M. B and K.D.M.

Supplementary material

427_2009_284_MOESM1_ESM.avi (179 kb)
S1 Confocal stacks of the actub-stained embryo (red) depicted in Fig. 4a, b′′′. Note the high number of nuclei at the anterior pole (sytox, green). In the first focal planes, there is strong background fluorescence (green) because of the eggshell. (AVI 179 kb)
427_2009_284_MOESM2_ESM.avi (179 kb)
S2 Confocal stacks of a tyrtub-stained embryo (red). Although there is some background fluorescence because of a fold in the eggshell, there is a clear anterior staining pattern. All the other nuclei of the embryo are stained by sytox (green). (AVI 179 kb)


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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Maxime Willems
    • 1
    Email author
  • Mieke Boone
    • 1
  • Marjolein Couvreur
    • 1
  • Katrien De Mulder
    • 1
  • Jelka Van Ranst
    • 1
  • Tom Artois
    • 2
  • Gaëtan Borgonie
    • 1
  1. 1.Nematology Section, Department of BiologyGhent UniversityGhentBelgium
  2. 2.Centre for Environmental Sciences, Research Group Biodiversity Phylogeny and Population StudiesHasselt UniversityDiepenbeekBelgium

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