Cite as: Cold Spring Harb. Protoc.; 2010; doi:10.1101/pdb.prot5356

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Mounting, Embedding, and Sectioning of Early Rabbit Embryos

Bernd Püschel1, Eva Bitzer2, Martin Blum2, and Christoph Viebahn1,3

1 Department of Anatomy and Embryology, University of Göttingen, 37075 Göttingen, Germany
2 Department of Zoology, University of Hohenheim, 70593 Stuttgart, Germany

3Corresponding author (cviebah{at}gwdg.de).


INTRODUCTION

This protocol describes various methods for visualizing cellular labeling in early rabbit embryos at high resolution using stereomicroscopy, compound light microscopy, or electron microscopy. Embedding media are chosen according to the level of resolution needed: For low-magnification stereomicroscopy, optimal spreading of large, flat, thin embryonic discs is achieved by mounting embryos in Mowiol on regular microscope slides. Gelatin/BSA embedding is useful for quickly cutting a complete series of thick (e.g., 30-µm) sections in defined planes from large (e.g., somite stage) paraformaldehyde-fixed embryos prelabeled by immunofluorescence or in situ hybridization. Higher cellular and subcellular resolution of such specimens usually requires embedding in Technovit, a hydrophilic methacrylate-based resin suited for light microscopic analysis of 5-µm serial sections. Araldite is a translucent, hydrophobic epoxy-based resin used for serial semi-thin (1-µm) and ultrathin (80-nm) sections amenable to consecutive (and correlative) light and electron microscopic analysis. Specimens to be embedded in Araldite must be much harder than those embedded in hydrophilic resins, requiring at least 1% glutaraldehyde in the primary fixative. Unfortunately, strong cross-linking by glutaraldehyde prevents the use of antibodies/nucleotide probes post-fixation; glutaraldehyde autofluorescence also thwarts the use fluorescent stains. Finally, semi-thin cryosections can be cut from unfixed or paraformaldehyde-fixed specimens embedded in "tissue freezing medium," offering the opportunity for highly sensitive light microscopic analysis of antigen distribution. Here, subcellular structures can be identified up to the level of individual mitochondria; greater resolution can be obtained by correlating such samples with similar specimens analyzed using other techniques.


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Related Article

The Rabbit (Oryctolagus cuniculus): A Model for Mammalian Reproduction and Early Embryology
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Cold Spring Harb Protoc 2010: 139. [Abstract] [Full Text]

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 Cold Spring Harb ProtocHome page
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