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1Interdisciplinary Center for Neurosciences, Institute of Anatomy and Cell Biology, University of Heidelberg
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We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.
Brachmann, I., Tucker, K. L. Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth. J. Vis. Exp. (49), e2309, doi:10.3791/2309 (2011).
For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue.
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.
Part 1: Preparing for slicing and culturing.
Part 2: Embryo embedding.
Part 3: Slicing procedure.
Part 4: Imaging spinal nerve outgrowth at the microscope.
Figure 1 shows an imaging series depicting the spinal nerve outgrowth during 20 hours of culture with 4x and 10x objectives.
Figure 1 Imaging series of spinal nerve outgrowth in a transverse slice of a homozygous tauGPF embryo. * = motor neurons of the ventral spinal cord, arrow = DRG. The dorsal (D)-ventral (V) axis of the slice is indicated. Scalebars: 200 μm.
In an extensive comparison of methods to prepare embryonic slice cultures of mid-gestation mouse embryos (E10 - E12), we have observed that a vibratome produces without question the most reliable results with respect to both the overall viability of the cultures and the reproducibility of the nerve outgrowth patterns. In contrast, slices prepared using a McIlwain tissue chopper3 proved to be completely inviable. We originally employed a guillotine method4, in which an entire litter of embryos could be simultaneously prepared by slicing with tungsten wires wrapped serially around a cutting grate to produce 400-μm sections1. Although this technique had the advantage of speed of preparation, it yielded at most one viable section per embryo and showed a large amount of variability in outgrowth parameters. For these reasons, we feel that a vibratome-based method is the superior choice despite the longer time in preparation. Although this solution by necessity requires a vibratome, the results are vastly superior to what has been achieved through other "low-tech" approaches, and thus justifies the investment.
The authors acknowledge the original source for the idea to perform slice culture upon mouse embryos5. We would like to acknowledge Joachim Kirsch for generous scientific support and Anna Degen for acting as our gofer during the filming. This work was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft: Sonderforschungsbereich 488, Teilprojekt B7/B9) and the University of Heidelberg (Excellence Cluster Cellular Networks).
|HBSS 10x||GIBCO, by Life Technologies||14180|
|Dissection tools||Fine Science Tools||various|
|Whatmann paper||Whatman, GE Healthcare||3030917|
|Shortened firepolished pipettes|
|DMEM||GIBCO, by Life Technologies||41966|
|FBS||GIBCO, by Life Technologies||10270-106|
|Pen Strep||GIBCO, by Life Technologies||15140|
|L-glutamine 100x||GIBCO, by Life Technologies||25030|
|Vibratome||Microm International||HM 650 V|
|Fluorescent microscope||Olympus Corporation||BX61WI|
|analySIS||Soft Imaging System|
|Millicell-CM inserts||EMD Millipore||PICMORG 50|
|10 cm culture plates||Greiner Bio-One||633171|
|LOCTITE 406||Henkel Corp||142580|
|Razor blades||Thermo Fisher Scientific, Inc.||none|
|Dissecting microscope||Nikon Instruments||SMZ800|
|HEPES||Carl Roth Gmbh||9105.2|
|x4 objective||Olympus Corporation||PL series|
|x10 objective||Olympus Corporation||UPLFL –PH series|
|CCD camera||Soft Imaging System||SIS F-View II|
|Equipment for heated chamber||Leica Microsystems||CTI-Controller 3700 and incubator S #11531171|
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