Use of freeze-cracking in ontogenetic research in Macrostomum lignano (Macrostomida, Rhabditophora)
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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.
KeywordsPlatyhelminthes 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.
- Gremigni V, Falleni A (1992) Mechanisms of shell-granule and yolk production in oocytes and vitellocytes of Platyhelminthes–Turbellaria. Anim Biol 1:29–37Google Scholar
- Ladurner P, Egger B, De Mulder K, Pfister D, Kuales G, Salvenmoser W, Schärer L (2008) The stem cell system of the basal flatworm Macrostomum lignano. In: Bosch TCG (ed) Stem cells: from Hydra to Man. Springer, The Netherlands, pp 75–95Google Scholar
- Pfister D, De Mulder K, Philipp I, Kuales G, Hrouda M, Eichberger P, Borgonie G, Hartenstein V, Ladurner P (2007) The exceptional stem cell system of Macrostomum lignano: screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation. Front Zool 4:9PubMedCrossRefGoogle Scholar
- Rieger R, Gehlen M, Hazpruhnar G, Homlund M, Legniti A, Salvenmoster W, Tyler S (1988) Laboratory cultures of marine Macrostomida (Turbellaria). Fortschr Zool 36:525Google Scholar
- Rieger RM, Tyler S, Smith JPS III, Rieger GE (1991b) Platyhelminthes: Turbellaria. In: Harrison FW, Bogitsh BJ (eds) Microscopic anatomy of invertebrates. Vol. 3. Platyhelminthes and Nemertinea. Wiley-Liss, New York, pp 7–140Google Scholar