We thank Linda Hildegard Bergersen and Magnar Bj&#248;r&#229;s for access to cell lab and equipment, Janelia Molecular Biology Shared Resource staff for plasmid and virus production and Koen Vervaeke for assistance with laser power measurements. This work was funded by the Norwegian Health Association, the Norwegian Research Council and the microscopy equipment was funded by Norbrain.

The procedures described here have been approved by the Norwegian Animal Research Authority. Suppliers and catalog numbers for the consumables and other required equipment are available in the materials list at the end of the document.
1. Preparation of Organotypic Slices
NOTE: This recipe uses two mouse pups at postnatal day 7-9 (p7-9), which yield 24 organotypic slices divided on two six-well culture dishes. Unless stated otherwise, all procedures should be done in ...

<ol>
	<li>Barry, C., Pearson, C., Barbarese, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+organization+of+oligodendrocyte+processes+during+development+in+culture+and+in+vivo.">Morphological organization of oligodendrocyte processes during development in culture and in vivo.</a> Dev. Neursosci. 18, 233-242 (1996).</li><li>Simpson, P. B., Mehotra, S., Lange, G. D., Russell, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+density+distribution+of+endoplasmic+reticulum+proteins+and+mitochondria+at+specialized+Ca2++release+sites+in+oligodendrocyte+processes.">High density distribution of endoplasmic reticulum proteins and mitochondria at specialized Ca2+ release sites in oligodendrocyte processes.</a> J. Biol. Chem. 272, 22654-22661 (1997).</li><li>Sterky, F. H., Lee, S., Wibom, R., Olson, L., Larsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+mitochondrial+transport+and+Parkin-independent+degeneration+of+respiratory+chain-deficient+dopamine+neurons+in+vivo.">Impaired mitochondrial transport and Parkin-independent degeneration of respiratory chain-deficient dopamine neurons in vivo.</a> Proc. Natl. Acad. Sci. U. S. A. 1080, 12937-12942 (2011).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J. Neurosci. Methods. 37, 173-182 (1991).</li><li>De Simoni, A., Yu, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+organotypic+hippocampal+slice+cultures:+interface+method.">Preparation of organotypic hippocampal slice cultures: interface method.</a> Nat. Protoc. 1, 1439-1445 (2006).</li><li>Lim, H., 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=Label-free+imaging+of+Schwann+cell+myelination+by+third+harmonic+generation+microscopy.">Label-free imaging of Schwann cell myelination by third harmonic generation microscopy.</a> Proc. Natl. Acad. Sci. U. S. A. 111, 18025-18030 (2014).</li><li>Farrar, M. J., Wise, F. W., Fetcho, J. R., Schaffer, C. B. <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+imaging+of+myelin+in+the+vertebrate+central+nervous+system+using+third+harmonic+generation+microscopy.">In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy.</a> Biophys. J. 100, 1362-1371 (2011).</li><li>Fu, Y., Wang, H., Huff, T. B., Shi, R., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+anti-Stokes+Raman+scattering+imaging+of+myelin+degradation+reveals+a+calcium-dependent+pathway+in+lyso-PtdCho-induced+demyelination.">Coherent anti-Stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination.</a> J. Neurosci. Res. 85, 2870-2881 (2007).</li><li>Schain, A. J., Hill, R. A., Grutzendler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+in+vivo+imaging+of+myelinated+axons+in+health+and+disease+with+spectral+confocal+reflectance+microscopy.">Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy.</a> Nat. Med. 20, 443-449 (2014).</li><li>Shin, J. H., Yue, Y., Duan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+viral+vector+production+and+purification.">Recombinant adeno-associated viral vector production and purification.</a> Methods Mol. Biol. 798, 267-284 (2012).</li><li>Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligonucleotide-directed+mutagenesis+without+phenotypic+selection.">Oligonucleotide-directed mutagenesis without phenotypic selection.</a> Curr. Prot. Neurosci. , 4.10.1-4.10.6 (2001).</li><li>Gow, A., Friedrich, V. L., Lazzarini, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+basic+protein+gene+contains+separate+enhancers+for+oligodendrocyte+and+Schwann+cell+expression.">Myelin basic protein gene contains separate enhancers for oligodendrocyte and Schwann cell expression.</a> J. Cell Biol. 119, 605-616 (1992).</li><li>Rinholm, J. E., 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=Movement+and+structure+of+mitochondria+in+oligodendrocytes+and+their+myelin+sheaths.">Movement and structure of mitochondria in oligodendrocytes and their myelin sheaths.</a> Glia. 64, 810-825 (2016).</li><li>Rintoul, G. L., Filiano, A. J., Brocard, J. B., Kress, G. J., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+decreases+mitochondrial+size+and+movement+in+primary+forebrain+neurons.">Glutamate decreases mitochondrial size and movement in primary forebrain neurons.</a> J. Neurosci. 23, 7881-7888 (2003).</li><li>Jackson, J. G., O'Donnell, J. C., Takano, H., Coulter, D. A., Robinson, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+and+glutamate+uptake+decrease+mitochondrial+mobility+in+astrocytes+and+position+mitochondria+near+glutamate+transporters.">Neuronal activity and glutamate uptake decrease mitochondrial mobility in astrocytes and position mitochondria near glutamate transporters.</a> J. Neurosci. 34, 1613-1624 (2014).</li><li>Macaskill, A. F., 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=Miro1+is+a+calcium+sensor+for+glutamate+receptor-dependent+localization+of+mitochondria+at+synapses.">Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses.</a> Neuron. 61, 541-555 (2009).</li><li>Karadottir, R., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+patch-clamping+of+cells+in+brain+slices+with+immunocytochemical+labeling+to+define+cell+type+and+developmental+stage.">Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage.</a> Nat. Protoc. 1, 1977-1986 (2006).</li><li>Rinholm, J. E., 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=Regulation+of+oligodendrocyte+development+and+myelination+by+glucose+and+lactate.">Regulation of oligodendrocyte development and myelination by glucose and lactate.</a> J. Neurosci. 31, 538-548 (2011).</li><li>Davison, A. N., Dobbing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+as+a+vulnerable+period+in+brain+development.">Myelination as a vulnerable period in brain development.</a> Br. Med. Bull. 22, 40-44 (1966).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Noraberg, J., Kristensen, B. W., Zimmer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+for+neuronal+degeneration+in+organotypic+slice+cultures.">Markers for neuronal degeneration in organotypic slice cultures.</a> Brain Res. Protoc. 3, 278-290 (1999).</li><li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> J. Neurosci. 30, 6171-6177 (2010).</li><li>Pignataro, D., 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=Adeno-Associated+Viral+Vectors+Serotype+8+for+Cell-Specific+Delivery+of+Therapeutic+Genes+in+the+Central+Nervous+System.">Adeno-Associated Viral Vectors Serotype 8 for Cell-Specific Delivery of Therapeutic Genes in the Central Nervous System.</a> Front. Neuroanat. 11 (2), (2017).</li><li>Hutson, T. H., Verhaagen, J., Yanez-Munoz, R. J., Moon, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticospinal+tract+transduction:+a+comparison+of+seven+adeno-associated+viral+vector+serotypes+and+a+non-integrating+lentiviral+vector.">Corticospinal tract transduction: a comparison of seven adeno-associated viral vector serotypes and a non-integrating lentiviral vector.</a> Gene Ther. 19, 49-60 (2012).</li><li>Neumann, S., Campbell, G. E., Szpankowski, L., Goldstein, L. S. B., Encalada, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+composition+of+molecular+motors+on+moving+axonal+cargo+using+cargo+mapping+analysis.">Characterizing the composition of molecular motors on moving axonal cargo using cargo mapping analysis.</a> J. Vis. Exp. (92), e52029 (2014).</li></ol>

The authors declare no conflicts of interest.

The protocol for making organotypic cultures described here is a modified version18 of the protocol described by De Simoni and Yu (2006)5. The most important changes have been outlined below. Tris buffer is added to the culture medium, which improves the survival of the slices when outside of the incubator during viral transduction and changing of the cell medium. The sterilization procedure for confetti is also changed. While other protocols sterilize confetti by autoclavi...

The brain&#39;s white matter is composed of nerve cell axons wrapped in myelin, a specialized extended plasma membrane formed by oligodendrocytes. Myelin is required for fast and reliable action potential propagation and long-term survival of myelinated axons, and a loss of myelin can cause neurological dysfunction. Despite their importance, the properties of oligodendrocytes are less known compared with neurons and astrocytes. Consequently, fewer tools have been developed for studying oligodendrocytes.
Live imaging of cell organelles such as mitochondria, endoplasmatic reticulum (ER) or different vesicular structures can be useful to study dynamic changes in the organelles over time. Traditionally, imaging of living oligodendrocytes has been performed in monocultures1,2. However, oligodendrocytes in monoculture do not display compact myelin, and organotypic or acute brain slices may, therefore, be a better option when studying localization and movement of organelles. Localization of small organelles and proteins in the myelin sheath can be challenging due to the short distance between the myelinated axon and the surrounding myelin sheath. Thus, light microscopic immunostaining procedures alone do not have the spatial resolution to discriminate between organelles in the myelin sheath and those in the myelinated axon. This can be solved by viral transduction with genes for organelle-targeted fluorescent proteins driven by cell type-specific promoters. The advantages are a cell-specific and sparse expression, which enables accurate assessment of organelle localization and dynamics. Transgenic animals can also be used to achieve such an organelle-targeted cell-specific expression3. However, the production and maintenance of transgenic animals is expensive and usually does not offer the sparse expression that can be achieved by viral methods.
The method described here uses viral transduction of oligodendrocytes with mitochondrial-targeted fluorescent proteins (dsred or green fluorescent protein, GFP) driven by the myelin basic protein promoter (MBP-mito-dsred or MBP-mito-GFP) to visualize oligodendrocyte mitochondria in organotypic brain slices. In addition, expression of another fluorescent protein in the cytoplasm (either GFP used together with mito-dsred or tdtomato used with mito-GFP) is used to enable visualization of cell morphology, including the cytoplasmic compartments of the myelin sheath. The protocol includes the procedure for making organotypic brain slices (a modified version of the protocol described by De Simoni and Yu, 20064,5). We then describe the time-lapse imaging procedure for studying mitochondrial movement. This procedure uses an upright confocal microscope with a continuous exchange of imaging medium, a setup that enables easy application of drugs or other medium changes during imaging. The time-lapse imaging procedure can be performed on any confocal microscope, with some extra equipment for maintaining living slices as described below. The protocol also contains several tips to optimize imaging and reduce phototoxicity.
Lastly, a quick and simple way to visualize unstained myelin by Confocal Reflectance microscopy (CoRe) is described. This can be useful to identify the myelin sheath during live imaging. In recent years, several techniques have been developed to image myelin without any staining required, but most of these require specific equipment and expertise6,7,8. The procedure described here uses the reflective properties of the myelin sheath and is a simplified single-excitation wavelength version of Spectral Confocal Reflectance microscopy (SCoRe, in which several laser wavelengths are combined to visualize myelin)9. CoRe can be done on any confocal microscope that has a 488 nm laser and a 470 - 500 nm bandpass emission filter or a tunable emission filter.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="36">
			<td height="36" style="height:36px;width:167px;">Agarose&#160;</td>
			<td style="width:139px;">Sigma&#160;</td>
			<td style="width:125px;">A9539</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:167px;">BD Microlance 19G</td>
			<td style="width:139px;">BD</td>
			<td style="width:125px;">301500</td>
			<td style="width:171px;">Needles used for in- and outlet of bath</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:167px;">Bioxide gas</td>
			<td style="width:139px;">AGA</td>
			<td style="width:125px;">105701</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:167px;">Brand pipette bulbs</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">Z615927</td>
			<td style="width:171px;">Pipette bulbs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:167px;">Bunsen burner (Liquid propane burner)</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">89038-530</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:167px;">Cable assembly for heater controllers</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:125px;">64-0106&#160;</td>
			<td style="width:171px;">Temperature controller - thermometer part</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:167px;">CaCl2</td>
			<td style="width:139px;">Fluka</td>
			<td style="width:125px;">21100</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:167px;">CO2&#160;</td>
			<td style="width:139px;">AGA</td>
			<td style="width:125px;">100309</td>
			<td style="width:171px;">CO2 for incubator</td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;width:167px;">Cover glass, square Corning</td>
			<td style="width:139px;">Thermo&#160; Fischer Scientific</td>
			<td style="width:125px;">13206778</td>
			<td style="width:171px;">To attach under bath for live imaging. Seal with glue or petrolium jelly.</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:167px;">D-(+)-Glucose</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">G7021</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:167px;">Delicate forceps</td>
			<td style="width:139px;">Finescience</td>
			<td style="width:125px;">11063-07</td>
			<td style="width:171px;">For dissection</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:167px;">Diamond scriber pen</td>
			<td style="width:139px;">Ted Pella Inc.</td>
			<td style="width:125px;">54463</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:167px;">Disposable Glass Pasteur Pipettes 230 mm</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">612-1702</td>
			<td style="width:171px;">Glass pipettes</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Double edge stainless steel razor blade</td>
			<td style="width:139px;">Electron Microscopy Sciences</td>
			<td style="width:125px;">#7200</td>
			<td style="width:171px;">Razor blade for vibratome</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:167px;">Earle&#39;s Balanced Salt Solution (EBSS)</td>
			<td style="width:139px;">Gibco-Invitrogen</td>
			<td style="width:125px;">24010-043</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Filter paper circles</td>
			<td style="width:139px;">Schleicher &#38; Schuell</td>
			<td style="width:125px;">300,220</td>
			<td style="width:171px;">Filter paper used for filtration of PFA</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Fun tack</td>
			<td style="width:139px;">Loctite</td>
			<td style="width:125px;">1270884</td>
			<td style="width:171px;">Use to connect/adjust position of in- and outlets in bath</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:167px;">Hand towel C-Fold 2</td>
			<td style="width:139px;">Katrin</td>
			<td style="width:125px;">344388</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:167px;">Harp, Flat for RC-41 Chamber,</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:125px;">&#160;64-1418</td>
			<td style="width:171px;">Harp to hold down confetti in bath. Cut off strings before use with organotypic slices. 1.5 mm, 13mm, SHD-41/15</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:167px;">HEPES, FW: 260.3</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">H-7006</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:167px;">Holten LaminAir, Model 1.2</td>
			<td style="width:139px;">Heto-Holten</td>
			<td style="width:125px;">96004000M</td>
			<td style="width:171px;">Laminar flow hood</td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:167px;">Horse serum, heat inactivated</td>
			<td style="width:139px;">Gibco-Invitrogen</td>
			<td style="width:125px;">26050-088</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:167px;">KCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">P9541</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:167px;">LCR Membrane, PTFE,&#160;</td>
			<td style="width:139px;">Millipore</td>
			<td style="width:125px;">FHLC0130</td>
			<td style="width:171px;">Confetti&#160;</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:167px;">Leica VT1200</td>
			<td style="width:139px;">Leica</td>
			<td style="width:125px;">14048142065</td>
			<td style="width:171px;">Vibratome</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:167px;">MEM-Glutamax with HEPES</td>
			<td style="width:139px;">Thermo&#160; Fischer Scientific</td>
			<td style="width:125px;">42360024</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:167px;">MgCl2</td>
			<td style="width:139px;">R.P. Normapur</td>
			<td style="width:125px;">25 108.295</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Micro Spoon Heyman Type B</td>
			<td style="width:139px;">Electron Microscopy Sciences</td>
			<td style="width:125px;">62411-B</td>
			<td style="width:171px;">Small, rounded spatula with sharpened end for dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Millex-GP filter unit</td>
			<td style="width:139px;">Millipore</td>
			<td style="width:125px;">SLGPM33RA</td>
			<td style="width:171px;">Syringe filter unit</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:167px;">Millicell cell culture insert, 30 mm</td>
			<td style="width:139px;">Millipore</td>
			<td style="width:125px;">PICM03050</td>
			<td style="width:171px;">Cell culture inserts</td>
		</tr>
		<tr height="90">
			<td height="90" style="height:90px;width:167px;">Minipuls 3 Speed Control Module</td>
			<td style="width:139px;">GILSON</td>
			<td style="width:125px;">F155001</td>
			<td style="width:171px;">Peristaltic pump for live imaging - Control module part (connect to two-cannel head)</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:167px;">Na2HPO4</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">S7907</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:167px;">NaCl</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">S9888</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:167px;">NaH2PO4</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">S8282</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:167px;">NaHCO3</td>
			<td style="width:139px;">Fluka</td>
			<td style="width:125px;">71628</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:167px;">Nunclon Delta Surface</td>
			<td style="width:139px;">Thermo&#160; Fischer Scientific</td>
			<td style="width:125px;">140675</td>
			<td style="width:171px;">Culture plate</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:167px;">Nystatin Suspension</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">N1638</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="93">
			<td height="93" style="height:93px;width:167px;">Objective W &#34;Plan-Apochromat&#34; 40x/1.0 DIC&#160;</td>
			<td style="width:139px;">Zeiss</td>
			<td style="width:125px;">441452-9900-000&#160;</td>
			<td style="width:171px;">Water immersion objective used for live imaging. (WD=2.5mm), VIS-IR</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:167px;">Parafilm</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">291-1211</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Paraformaldehyde, granular</td>
			<td style="width:139px;">Electron Microscopy Sciences</td>
			<td style="width:125px;">#19208</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:167px;">PC-R perfusion chamber</td>
			<td style="width:139px;">SiSkiYou</td>
			<td style="width:125px;">&#160;15280000E</td>
			<td style="width:171px;">Bath for live imaging</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:167px;">Penicillin-Streptomycin, liquid</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:125px;">15070-063</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:167px;">Petri dish 140 mm&#160;</td>
			<td style="width:139px;">Heger</td>
			<td style="width:125px;">1075</td>
			<td style="width:171px;">Large Petri dish&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:167px;">Petri dish 92x16 mm&#160;</td>
			<td style="width:139px;">Sarstedt&#160;</td>
			<td style="width:125px;">82.1473</td>
			<td style="width:171px;">Medium Petri dish&#160;</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:167px;">Petridish 55x14,2 mm</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">391-0868</td>
			<td style="width:171px;">Small Petri dish</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:167px;">Phosphate buffered saline (PBS)</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">P4417</td>
			<td style="width:171px;">PBS tablets</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">R2 Two Channel Head&#160;</td>
			<td style="width:139px;">GILSON</td>
			<td style="width:125px;">F117800</td>
			<td style="width:171px;">Peristaltic pump for live imaging - Two channel head part (requires control module)</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:167px;">Round/Flat Spatulas, Stainless Steel</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">82027-528</td>
			<td style="width:171px;">Large spatula for dissection</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Sand paper</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">MMMA63119</td>
			<td style="width:171px;">Optional, for smoothing broken glass pipettes</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Scissors,&#160; 17,5 cm</td>
			<td style="width:139px;">Finescience</td>
			<td style="width:125px;">14130-17</td>
			<td style="width:171px;">Large scissors for dissection</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Scissors, 8,5&#160;</td>
			<td style="width:139px;">Finescience</td>
			<td style="width:125px;">14084-08</td>
			<td style="width:171px;">Small, sharp scissors for&#160; dissection</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Single edge, gem blade</td>
			<td style="width:139px;">Electron Microscopy Sciences</td>
			<td style="width:125px;">#71972</td>
			<td style="width:171px;">Single edge razor blade</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:167px;">Single inline solution heater SH-27B</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:125px;">64-0102</td>
			<td style="width:171px;">Temperature controller - heater part</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:167px;">Steritop-GP Filter unit, 500 ml , 45mm</td>
			<td style="width:139px;">Millipore</td>
			<td style="width:125px;">SCGPT05RE</td>
			<td style="width:171px;">Filter to sterilize solutions</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:167px;">Super glue precision</td>
			<td style="width:139px;">Loctite</td>
			<td style="width:125px;">1577386</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:167px;">Surgical scalpel blade no. 22</td>
			<td style="width:139px;">Swann Morton Ltd.</td>
			<td style="width:125px;">209</td>
			<td style="width:171px;">Rounded scalpel blade</td>
		</tr>
		<tr height="113">
			<td height="113" style="height:113px;width:167px;">Temperature controller TC324B</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:125px;">64-0100</td>
			<td style="width:171px;">Temperature controller for live imaging (requires solution heater and cable assembly)</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:167px;">Trizma base</td>
			<td style="width:139px;">Sigma&#160;</td>
			<td style="width:125px;">T1503</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:167px;">Trizma HCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">T3253</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:167px;">Water jacketed incubator series II</td>
			<td style="width:139px;">Forma Scientific</td>
			<td style="width:125px;">78653-2882</td>
			<td style="width:171px;">Incubator</td>
		</tr>
	</tbody>
</table>

visualization and live imaging of oligodendrocyte organelles in organotypic brain slices using adeno-associated virus and confocal microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Organotypic brain slices that were cultured and transduced as described above showed a sparse distribution of cortical oligodendrocytes expressing mito_dsred and GFP. Immunostaining with antibodies against Olig2 and MBP confirmed that the expression was specific to oligodendrocytes (Figure 1).
For live imaging, transduced oligodendrocytes were recognized by their characteristic morphology of several...

Watch this Scientific Journal Video about Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy at JoVE.com

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

The overall goal of this viral transduction and time-lapse recording is to visualize movement of oligodendrocyte mitochondria in organotypic brain slices.This method can help answer key questions in the oligodendrocyte field.Such as how the oligodendrocytes and their organelles respond to changes in the local environment.The main advantage of this technique is that it gives detailed information about oligodendrocyte structure and organelle dynamics in a preparation where all the different brain cells are present and oligodendrocytes produce compact myelin.Transduce oligodendrocytes in slice cultures at day seven in vitro.First draw up 1.3 microliters of diluted AAV per slice.Making sure the outside of the pipette tip is dry, hold pipette above the cortex as close as possible without touching the slice with the pipette tip and slowly push solution out of the pipette.When the solution touches the slice, move the pipette tip over the cortex to spread the solution over the whole cortex area.Once all the slices in one dish have been transduced, return the dish to the incubator and then repeat the process on the other dishes.Examine the slices for expression of fluorescent markers and image the slices when the expression levels of the fluorescent markers are sufficient and the slices look healthy.Begin setting up the imaging bath by using modeling clay to attach syringe needles bent at a 45

visualization-live-imaging-oligodendrocyte-organelles-organotypic

Research

JoVE Journal

Neuroscience

10.7K Görüntüleme.  University of Oslo. The overall goal of this viral transduction and time-lapse recording is to visualize movement of oligodendrocyte mitochondria in organotypic brain slices.This method can help answer key questions in the oligodendrocyte field.Such as how the oligodendrocytes and their organelles respond to changes in the local environment.The main advantage of this technique is that it gives detailed information about oligodendrocyte structure and organelle dynamics in a preparation where all the different bra...