Name: Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth (Video) | JoVE Journal
Uploaded: 2011-03-29T11:00:00.000+00:00
Duration: 5 min 42 s
Description: Learn organotypic slice culture of GFP-expressing mouse embryos for real-time imaging of peripheral nerve outgrowth. This method enables fluorescence microscopy studies of spinal nerve development in developmental neurobiology.

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5 minutes

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).

Part 1: Preparing for slicing and culturing.
<ol>
	<li>Prepare 10-cm tissue culture plates with slicing medium (DMEM, 25% 1x HBSS, 25% fetal bovine serum, 0.5% glucose, 1 mM glutamine, 2.5 mM HEPES, pH 7.3) and 3-cm Millicell-CM 0.4-μm culture membrane inserts and keep in incubator at 37°C and 5% CO2.</li>
	<li>Heat up 4% low melting point agarose in PBS in the microwave and keep it on heating plate so that it stays at approximately 37°C.</li>
	<li>Fill 10-cm bacteriological Petri dishes with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8mM KH2PO4) and place on ice.</li>
	<li>Set up microtome cooling device or ensure that buffer tray and cooling elements are stored in the freezer to pre-cool it.</li>
</ol>
Part 2: Embryo embedding.
<ol>
	<li>Dissect embryos from the uterus and examine them with an inverted fluorescent microscope to check for GFP expression.</li>
	<li>Place embryos on an inverted 10-cm Petri dish and orient them using Whatman paper strips to remove excessive PBS.</li>
	<li>Apply agarose on embryo to fix it in this position. Let agarose solidify.</li>
	<li>Limit the area surrounding the embryo by cutting with a razor blade.</li>
	<li>Rotate the embedded embryo onto its other side.</li>
	<li>Apply additional agarose on embryonic tissue to ensure that the embryo is completely embedded.</li>
	<li>Prepare agarose block with clean edges and mount on vibratome chuck using Loctite 406, a special glue similar to "Krazy Glue".</li>
</ol>
Part 3: Slicing procedure.
<ol>
	<li>Set up the precooled buffer tray and the cooling element.</li>
	<li>Insert the chuck with the glued tissue and add 1x HBSS (Ca2+-Mg2+-free HBSS, 10 mM HEPES buffer pH 7.3, 500 U/ml penicillin/streptomycin) until covered.</li>
	<li>Insert and fix a precleaned (70% Ethanol) microtome blade.</li>
	<li>Prepare 350 - 450 μm slices and transfer them using shortened glass pasteur pipettes into tissue culture plates kept on ice.</li>
	<li>Using a pair of forceps, carefully remove agarose from each slice and transfer to Millicell culture membranes. About 4 slices can be cultured on one membrane in a 10-cm tissue culture plate filled with 6 mL of culture medium.</li>
	<li>Incubate slices at 37°C and 5% CO2 (Culture medium: DMEM, 25% 1x HBSS, 25% fetal bovine serum, 0.5% glucose, 1mM glutamine, 2.5 mM HEPES, pH 7.3). If one performs time-lapse imaging series one should keep the volume of medium constant. In the case of timed imaging series for a short time frame it is sufficient to leave the medium as it is. For longer culture periods changes after 12-20 hours are recommended.</li>
</ol>
Part 4: Imaging spinal nerve outgrowth at the microscope.


<ol>
	<li>Image spinal nerves placing a 10-cm tissue culture plate, containing Millicell culture membranes with slices, under an upright fluorescent microscope.</li>
	<li>Label the orientation of Millicell culture membranes on microscope stage to position correctly during the next imaging time point.</li>
	<li>Image spinal nerve outgrowth using 4x (numerical aperture [NA] 0.1), 10x (NA 0.3), or 20x (NA 0.5) objectives.</li>
</ol>
Figure 1 shows an imaging series depicting the spinal nerve outgrowth during 20 hours of culture with 4x and 10x objectives.
<img src="/files/ftp_upload/2309/2309fig1.jpg" alt="Time-lapse embryonic nerve growth; microscope image; 4h, 14h, 24h; developmental analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> 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.

<ol><li>Tucker, K. L., Meyer, M., Barde, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotrophins+are+required+for+nerve+growth+during+development%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Neurotrophins are required for nerve growth during development.</a> Nat Neurosci. 4, 29-37 (2001).</li>
<li>Brachmann, I., Jakubick, V. C., Shaked, M., Unsicker, K., Tucker, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+slice+culture+system+for+the+imaging+of+nerve+development+in+embryonic+mouse%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">A simple slice culture system for the imaging of nerve development in embryonic mouse.</a> Dev Dyn. 236, 3514-3523 (2007).</li>
<li>Collingridge, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+brain+slice+preparation%3A+a+tribute+to+the+pioneer+Henry+McIlwain%5BTitle%5D)+AND+%22Journal+of+neuroscience+methods%22%5BJournal%5D)">The brain slice preparation: a tribute to the pioneer Henry McIlwain.</a> Journal of neuroscience methods. 59, 5-9 (1995).</li>
<li>Katz, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+circuitry+of+identified+projection+neurons+in+cat+visual+cortex+brain+slices%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Local circuitry of identified projection neurons in cat visual cortex brain slices.</a> J Neurosci. 7, 1223-1249 (1987).</li>
<li>Hotary, K. B., Landmesser, L. T., Tosney, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryo+slices%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">Embryo slices.</a> Methods Cell Biol. 51, 109-124 (1996).</li>
</ol>

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 ...

<table><tbody><tr><td>HBSS 10x</td><td>GIBCO, by Life Technologies</td><td>14180</td><td></td></tr><tr><td>Dissection tools</td><td>Fine Science Tools</td><td>various</td><td></td></tr><tr><td>L.M.P. agarose</td><td>Invitrogen</td><td>15517-022</td><td></td></tr><tr><td>Whatmann paper</td><td>Whatman, GE Healthcare</td><td>3030917</td><td></td></tr><tr><td>Shortened firepolished pipettes</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>GIBCO, by Life Technologies</td><td>41966</td><td></td></tr><tr><td>FBS</td><td>GIBCO, by Life Technologies</td><td>10270-106</td><td></td></tr><tr><td>Pen Strep</td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>L-glutamine 100x</td><td>GIBCO, by Life Technologies</td><td>25030</td><td></td></tr><tr><td>Vibratome</td><td>Microm International</td><td>HM 650 V</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Olympus Corporation</td><td>BX61WI</td><td></td></tr><tr><td>analySIS</td><td>Soft Imaging System</td><td></td><td></td></tr><tr><td>Millicell-CM inserts</td><td>EMD Millipore</td><td>PICMORG 50</td><td></td></tr><tr><td>10 cm culture plates</td><td>Greiner Bio-One</td><td>633171</td><td></td></tr><tr><td>LOCTITE 406</td><td>Henkel Corp</td><td>142580</td><td></td></tr><tr><td>Razor blades</td><td>Thermo Fisher Scientific, Inc.</td><td>none</td><td></td></tr><tr><td>Dissecting microscope</td><td>Nikon Instruments</td><td>SMZ800</td><td></td></tr><tr><td>HEPES</td><td>Carl Roth Gmbh</td><td>9105.2</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>x4 objective</td><td>Olympus Corporation</td><td>PL series</td><td></td></tr><tr><td>x10 objective</td><td>Olympus Corporation</td><td>UPLFL &amp;#8211;PH series</td><td></td></tr><tr><td>Filter</td><td>Olympus Corporation</td><td>U-MNIBA2</td><td></td></tr><tr><td>CCD camera</td><td>Soft Imaging System</td><td>SIS F-View II</td><td></td></tr><tr><td>Equipment for heated chamber</td><td>Leica Microsystems</td><td>CTI-Controller 3700 and incubator S #11531171</td><td></td></tr></tbody></table>

organotypic slice culture of gfp-expressing mouse embryos for real-time imaging of peripheral nerve outgrowth

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

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.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

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 ...

The overall goal of this procedure is to prepare organotypic transverse slices of the forelimb region of GFP expressing mouse embryos to image spinal nerve outgrowth. This is accomplished by first embedding the embryos in low melting point aros and limiting the area of tissue and aros to the region of interest. The aros block is then mounted for slicing.Then the embryos embedded in the block are sliced with the vibrator. The final step of the procedure is to remove the aros surrounding the slices and to transfer the slices into culture plates. The slices are then cultured and show outgrowth of peripheral nerves into the periphery, which can be monitored with fluorescence microscopy.The main advantage of this technique over existing methods like a guillotine or a tissue chopper, is that the slices are generated in a more controlled fashion and show improved viability in culture. Therefore, this method can help in the study of developmental neurobiology, such as looking at the guidance and the outgrowth of spinal nerves, demonstrating the procedure will be Isabel barman. Begin this procedure by preparing 10 centimeter tissue culture plates with six milliliters of culture medium and three centimeter milli cell cm, 0.4 micron culture membrane inserts.Keep them in the incubator at 37 degrees Celsius and 5%carbon dioxide. Next, heat up 4%low melting point aros in PBS in the microwave, and keep it on a heating plate so that it stays at approximately 37 degrees Celsius. Fill 10 centimeter bacteriological Petri dishes with PBS and place them on ice.Then set up the Viome cooling device. Store the buffer tray and cooling elements in the freezer until thoroughly frozen to perform embryo. Embedding first dissect embryos from the uterus.Examine them with an inverted fluorescent microscope and check for GFP expression. Next place embryos on an inverted 10 centimeter Petri dish using watman paper strips to orient them and remove excessive PBS. Apply aros on the embryo in order to fix it.In the oriented position, wait for the aros to solidify trim excessive aros and if desired tissue surrounding the middle portion of the embryo. Rotate the embedded embryo to the other side. Apply additional aros on the embryonic tissue to ensure that the embryo is completely embedded.Wait for the aros to solidify. Prepare embryo embedded aros block with clean edges. Finally, glue the sample on a viome chuck with Loctite 4 0 6.Set up the pre cooled buffer tray and the cooling elements. Place the chuck with the glued tissue on the viome. Add one X-H-B-S-S to the chuck until the tissue is covered.Insert a pre-cleaned viome blade in the blade holder. Next, prepare transverse slices with a thickness of about 350 to 450 microns. Each slices are cut using an HM six 50 V microtome.Transfer the slices to tissue culture plates with a shortened glass pasture pipette and keep them on ice. Using a pair of forceps, carefully remove aros from all the slices. Then transfer the slices to the milli cell culture membranes with an arrangement of four slices on each membrane to 10 centimeter culture plates filled with six milliliters of prewarm culture.Medium.Incubate the slices at 37 degrees Celsius and 5%carbon dioxide until the beginning of time-Lapse imaging and between imaging intervals. Place the tissue culture plate containing Millie cell culture membranes with slices under an upright fluorescent microscope and locate the spinal nerves image spinal nerve outgrowth using four x 10 x or 20 x objectives. Label the orientation of milli cell culture membranes on the microscope stage to reposition correctly during the next imaging time point in case one doesn't have a temperature and carbon dioxide stable.Environment shown here is an imaging series of spinal nerve outgrowth during 24 hours of culture in a transverse slice of a homozygous tau GFP embryo. The star indicates motor neurons of the ventral spinal cord and the arrow indicates DRG. The upper panel was imaged using a four X objective and the bottom panel with a 10 x objective.After watching this video, you should have a good understanding of how to prepare organotypic transverse slices of mouse embryos that can be used to image spinal nerve outgrowth.

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