We would like to thank Manuela Budak, Ljiljana Marinkovi and Kristina Wullimann for excellent technical assistance. We thank W. Macklin for providing Plp-GFP mice. T.M. is supported by the Institute of Advanced Studies (Technische Universit&auml;t M&uuml;nchen), by the Deutsche Forschungsgemeinschaft (DFG; Sonderforschungsbereich SFB 596), by the Alexander-von-Humboldt-Foundation and by the national funding agency ("Bundesministerium f&uuml;r Bildung und Forschung") in the frame of ERA-Net NEURON "iPSoALS" and "2-photon imaging". T.M. and M.B. are supported by the Center for Integrated Protein Science (Munich). P.M. was supported by the Graduate School of Technische Universit&auml;t M&uuml;nchen (TUM-GS).

1. Triangularis Sterni Explant (Figure 1)
Timing: 15 min.
<ol>
 <li>Prepare surgical instruments: 2 pairs of forceps, 1 pair of scissors, 1 pair of spring scissors, 1 tissue culture dish (10-cm). Pre-bubbled (minimum for 15 min) cooled (4 &deg;C) Ringer's solution with 5% CO2/ 95% O2. Fill 15-cm tissue culture dish with ice and cover it with metal plate. Prepare dissection microscope and place the 15-cm dish with ice under the dissection scope in order ...

<ol>
	<li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Mallon, B. S., Shick, H. E., Kidd, G. J., Macklin, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolipid+promoter+activity+distinguishes+two+populations+of+NG2-positive+cells+throughout+neonatal+cortical+development.">Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development.</a> J. Neurosci. 22, 876-885 (2002).</li><li>Brill, M. S., Lichtman, J. W., Thompson, W., Zuo, Y., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+constraints+dictate+glial+territories+at+murine+neuromuscular+junctions.">Spatial constraints dictate glial territories at murine neuromuscular junctions.</a> The Journal of cell biology. 195, 293-305 (2011).</li><li>Livet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450, 56-62 (2007).</li><li>Thompson, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+with+photoswitchable+GFP+in+Schwann+cells.">Transgenic mice with photoswitchable GFP in Schwann cells.</a> Society for Neuroscience. Program 241, (2008).</li><li>Williams, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+development+of+outer+retinal+synapses+in+the+absence+of+glial+contact.">In vivo development of outer retinal synapses in the absence of glial contact.</a> J. Neurosci. 30, 11951-11961 (2010).</li><li>Kerschensteiner, M., Reuter, M. S., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+imaging+of+motor+axon+dynamics+in+murine+triangularis+sterni+explants.">Ex vivo imaging of motor axon dynamics in murine triangularis sterni explants.</a> Nat. Protoc. 3, 1645-1653 (2008).</li><li>McArdle, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+the+triangularis+sterni+muscle+of+the+mouse+for+investigations+of+synaptic+phenomena.">Advantages of the triangularis sterni muscle of the mouse for investigations of synaptic phenomena.</a> J. Neurosci. Methods. 4, 109-115 (1981).</li><li>Marinkovi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+deficits+and+degeneration+can+evolve+independently+in+mouse+models+of+amyotrophic+lateral+sclerosis.">Axonal transport deficits and degeneration can evolve independently in mouse models of amyotrophic lateral sclerosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 4296-4301 (2012).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7, 27-41 (1998).</li><li>Brill, M. S., Lichtman, J. W., Thompson, W., Zuo, Y., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+constraints+dictate+glial+territories+at+murine+neuromuscular+junctions.">Spatial constraints dictate glial territories at murine neuromuscular junctions.</a> The Journal of cell biology. 195, 293-305 (2011).</li><li>Zuo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+expressed+in+mouse+transgenic+lines+mark+subsets+of+glia,+neurons,+macrophages,+and+dendritic+cells+for+vital+examination.">Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.</a> J. Neurosci. 24, 10999-11009 (2004).</li></ol>

The SC bleaching method presented here is simple, fast and versatile: (i) It enables revealing single SC morphology and dynamics by confocal microscopy based on a single transgene - which is a significant advantage when combining the approach e.g. with disease models that require additional alleles. (ii) The method is rapid, as from dissection to fixation it can be performed in half a day. (iii) The number of applications is broad, as it can be combined with other methods, such as cell ablations, imaging organel...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company (example)</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>70% (vol/vol) ethanol solution </td>
 <td>Roth</td>
 <td>T913.1</td>
 <td>in spray bottle</td>
 </tr>
 <tr>
 <td>isoflurane</td>
 <td>Forene, Abbott</td>
 <td></td>
 <td>any other approved anaesthetic can be used for lethal anaesthesia</td>
 </tr>
 <tr>
 <td>transgenic mice (Plp-GFP) </td>
 <td></td>
 <td></td>
 <td>to be obtained from cited sources</td>
 </tr>
 <tr>
 <td>dissection microscope with cold-light illumination </td>
 <td>Olympus SZ51</td>
 <td></td>
 <td>equipped with Schott KL 1500 LCD</td>
 </tr>
 <tr>
 <td>forceps #2</td>
 <td>Fine Science Tools </td>
 <td>11233-20</td>
 <td></td>
 </tr>
 <tr>
 <td>forceps #5</td>
 <td>Fine Science Tools</td>
 <td>11295-00</td>
 <td></td>
 </tr>
 <tr>
 <td>scissors, large medical</td>
 <td>Fine Science Tools</td>
 <td>14108-09 </td>
 <td></td>
 </tr>
 <tr>
 <td>scissors, small angled spring</td>
 <td>Fine Science Tools</td>
 <td>15033-09 </td>
 <td></td>
 </tr>
 <tr>
 <td>in-line heater</td>
 <td>Warner Instruments</td>
 <td>SF-28</td>
 <td></td>
 </tr>
 <tr>
 <td>heating ring for 3.5-cm dishes </td>
 <td>Warner Instruments</td>
 <td>64-0110 DH-35 </td>
 <td></td>
 </tr>
 <tr>
 <td>two channel temperature control system</td>
 <td>Warner Instruments</td>
 <td>TC-344B</td>
 <td></td>
 </tr>
 <tr>
 <td>superfusion pump system </td>
 <td></td>
 <td></td>
 <td>custom-built</td>
 </tr>
 <tr>
 <td>15-cm tissue culture dish </td>
 <td>Sarstedt</td>
 <td>83.1803.003</td>
 <td>filled with ice and covered by metal plate</td>
 </tr>
 <tr>
 <td>10-cm tissue culture dish </td>
 <td>Sarstedt</td>
 <td>83.1802.003</td>
 <td>with oxygenated Ringer's solution</td>
 </tr>
 <tr>
 <td>3.5-cm tissue culture dish </td>
 <td>Sarstedt</td>
 <td>83.1800.003</td>
 <td>filled with Sylgard polymer</td>
 </tr>
 <tr>
 <td>Sylgard polymer</td>
 <td>Dow Corning</td>
 <td>Sylgard 184 Silicone Elastomer Kit</td>
 <td></td>
 </tr>
 <tr>
 <td>confocal microscope </td>
 <td>Olympus</td>
 <td>FV1000</td>
 <td>with argon laser</td>
 </tr>
 <tr>
 <td>50-ml reaction tube</td>
 <td>Sarstedt</td>
 <td>62.547.254 PP</td>
 <td></td>
 </tr>
 <tr>
 <td>4% PFA in PBS</td>
 <td>Sigma</td>
 <td>P6148</td>
 <td></td>
 </tr>
 <tr>
 <td>cannula 0.5 mm x 25 mm</td>
 <td>Neolab</td>
 <td>E-1510</td>
 <td></td>
 </tr>
 <tr>
 <td>syringe 1 ml</td>
 <td>Neolab</td>
 <td>E-1496</td>
 <td></td>
 </tr>
 <tr>
 <td>synaptophysin antibody</td>
 <td>Invitrogen</td>
 <td>18-0130</td>
 <td>rabbit</td>
 </tr>
 <tr>
 <td>secondary antibody</td>
 <td>Invitrogen</td>
 <td>A-11012</td>
 <td>Alexa 594</td>
 </tr>
 <tr>
 <td>24-well plate</td>
 <td>Sarstedt</td>
 <td>83.1836</td>
 <td></td>
 </tr>
 <tr>
 <td>0.01 M PBS</td>
 <td>Sigma</td>
 <td>P4417</td>
 <td></td>
 </tr>
 <tr>
 <td>0.1 M glycine in PBS</td>
 <td>Roth</td>
 <td>3790.1</td>
 <td></td>
 </tr>
 <tr>
 <td>coverslips</td>
 <td>Neolab</td>
 <td>E-4132</td>
 <td></td>
 </tr>
 <tr>
 <td>slides</td>
 <td>Neolab</td>
 <td>E-4121</td>
 <td></td>
 </tr>
 <tr>
 <td>Vectashield</td>
 <td>Vector labs</td>
 <td>H-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>minutien pins &times;10</td>
 <td>Fine Science Tools</td>
 <td>26002-20 </td>
 <td>shortened to &lt; 4 mm</td>
 </tr>
 <tr>
 <td>&alpha;-Bungarotoxin coupled to Alexa 594</td>
 <td>Invitrogen (Molecular Probes) </td>
 <td>B-13423</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Blocking Solution</td>
 </tr>
 <tr>
 <td>0.01 M PBS</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>0.2% Triton-X100</td>
 <td>Roth </td>
 <td>3051.3</td>
 <td></td>
 </tr>
 <tr>
 <td>10% Normal Goat Serum</td>
 <td>Abcam </td>
 <td>ab7481 </td>
 <td></td>
 </tr>
 <tr>
 <td> 1% bovine serum albumin</td>
 <td>Sigma </td>
 <td>A7030</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Ringer 
 bubbled with 95% O2 /5% CO2</td>
 </tr>
 <tr>
 <td> 125 mM NaCl </td>
 <td>Sigma </td>
 <td>S7653 </td>
 <td></td>
 </tr>
 <tr>
 <td>2.5 mM KCl</td>
 <td>Sigma </td>
 <td>P9333 </td>
 <td></td>
 </tr>
 <tr>
 <td>1.25 mM NaH2PO4</td>
 <td>Sigma </td>
 <td>S8282 </td>
 <td></td>
 </tr>
 <tr>
 <td>26 mM NaHCO3</td>
 <td>Sigma </td>
 <td>S6297 </td>
 <td></td>
 </tr>
 <tr>
 <td>2 mM CaCl2</td>
 <td>Sigma </td>
 <td>21114 </td>
 <td></td>
 </tr>
 <tr>
 <td>1 mM MgCl2</td>
 <td>Sigma </td>
 <td>63020</td>
 <td></td>
 </tr>
 <tr>
 <td>20 mM glucose</td>
 <td>Sigma </td>
 <td>G7528 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Specific reagents and equipments.</td>
 </tr>
 </tbody>
 </table>

sequential photo-bleaching to delineate single schwann cells at the neuromuscular junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

An example of a triangularis sterni explant ready for imaging dish is shown in Figure 1G. This explant is particularly suitable for imaging NMJs ex vivo as the triangularis muscle consists only of a few layers of muscle fibers. This allows obtaining high resolution images from explants derived from transgenic mouse lines that highlight either SCs (Plp-GFP2) and/or axons (Thy1-XFP1). Critical factors for high quality imaging include: (i) avoiding touching t...

Watch this Scientific Journal Video about Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction at JoVE.com

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

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10.5K Views.  Technische Universität München. Visualizzare le singole celle di densamente tessuti confezionati, come le cellule di Schwann terminali (SC) a giunzioni neuromuscolari (NMJs), è una sfida. "Sequenziale foto-bleaching" permette di delineare SC singolo terminale, per esempio nel triangularis espianto muscolo Sterni, un comodo nervo-muscolo preparazione, dove sequenziale sbiancamento può essere combinato con time-lapse imaging e Post-hoc Immunostainings.