Name: Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining (Video) | JoVE
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Description: Watch this Scientific Journal Video about Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining at JoVE.com

This research was supported in part by the Intramural Research Program of the NIH, NIDCD to M.H. (DC000088)

All animal experiments and procedures were performed according to protocols approved by the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. All experimental protocols were approved by the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of ...

Stria Vascularis Microdissection for Single-Nucleus Sequencing or Immunostaining

<ol>
	<li>Bosher, S. K., Warren, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+electrochemistry+of+the+cochlear+endolymph+of+the+rat:+a+quantitative+study+of+its+electrical+potential+and+ionic+composition+as+determined+by+means+of+flame+spectrophotometry.">Observations on the electrochemistry of the cochlear endolymph of the rat: a quantitative study of its electrical potential and ionic composition as determined by means of flame spectrophotometry.</a> Proceedings of the Royal Society of London. Series B. Biological Sciences. 171 (1023), 227-247 (1968).</li><li>Patuzzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+flow+in+stria+vascularis+and+the+production+and+regulation+of+cochlear+endolymph+and+the+endolymphatic+potential.">Ion flow in stria vascularis and the production and regulation of cochlear endolymph and the endolymphatic potential.</a> Hearing Research. 277 (1-2), 4-19 (2011).</li><li>Wangemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K++cycling+and+the+endocochlear+potential.">K+ cycling and the endocochlear potential.</a> Hearing Research. 165 (1-2), 1-9 (2002).</li><li>Adachi, N., 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=The+mechanism+underlying+maintenance+of+the+endocochlear+potential+by+the+K++transport+system+in+fibrocytes+of+the+inner+ear.">The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear.</a> The Journal of Physiology. 591 (18), 4459-4472 (2013).</li><li>Hibino, H., Nin, F., Tsuzuki, C., Kurachi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+is+the+highly+positive+endocochlear+potential+formed?+The+specific+architecture+of+the+stria+vascularis+and+the+roles+of+the+ion-transport+apparatus.">How is the highly positive endocochlear potential formed? The specific architecture of the stria vascularis and the roles of the ion-transport apparatus.</a> Pflugers Archiv. 459 (4), 521-533 (2010).</li><li>Lang, F., Vallon, V., Knipper, M., Wangemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+significance+of+channels+and+transporters+expressed+in+the+inner+ear+and+kidney.">Functional significance of channels and transporters expressed in the inner ear and kidney.</a> American Journal of Physiology. Cell Physiology. 293 (4), C1187-C1208 (2007).</li><li>Liu, W., Schrott-Fischer, A., Glueckert, R., Benav, H., Rask-Andersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+&amp;#34;cochlear+battery&amp;#34;-claudin-11+barrier+and+ion+transport+proteins+in+the+lateral+wall+of+the+cochlea.">The human &amp;#34;cochlear battery&amp;#34;-claudin-11 barrier and ion transport proteins in the lateral wall of the cochlea.</a> Frontiers in Molecular Neuroscience. 10, 239 (2017).</li><li>Marcus, D. C., Wu, T., Wangemann, P., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNJ10+(Kir4.1)+potassium+channel+knockout+abolishes+endocochlear+potential.">KCNJ10 (Kir4.1) potassium channel knockout abolishes endocochlear potential.</a> American Journal of Physiology. Cell Physiology. 282 (2), C403-C407 (2002).</li><li>Spicer, S. S., Schulte, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+inner+ear+fibrocytes+according+to+their+ion+transport+related+activity.">Differentiation of inner ear fibrocytes according to their ion transport related activity.</a> Hearing Research. 56 (1-2), 53-64 (1991).</li><li>Wangemann, P., Liu, J., Marcus, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+transport+mechanisms+responsible+for+K++secretion+and+the+transepithelial+voltage+across+marginal+cells+of+stria+vascularis+in+vitro.">Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro.</a> Hearing Research. 84 (1-2), 19-29 (1995).</li><li>Yoshida, T., 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=The+unique+ion+permeability+profile+of+cochlear+fibrocytes+and+its+contribution+to+establishing+their+positive+resting+membrane+potential.">The unique ion permeability profile of cochlear fibrocytes and its contribution to establishing their positive resting membrane potential.</a> Pflugers Archiv. 468 (9), 1609-1619 (2016).</li><li>Johns, J. D., Adadey, S. M., Hoa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+stria+vascularis+in+neglected+otologic+disease.">The role of the stria vascularis in neglected otologic disease.</a> Hearing Research. 428, 108682 (2023).</li><li>Kim, J., Ricci, A. J. <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+real-time+imaging+reveals+megalin+as+the+aminoglycoside+gentamicin+transporter+into+cochlea+whose+inhibition+is+otoprotective.">In vivo real-time imaging reveals megalin as the aminoglycoside gentamicin transporter into cochlea whose inhibition is otoprotective.</a> Proceedings of the National Academy of Sciences. 119 (9), e2117846119 (2022).</li><li>Zdebik, A. A., Wangemann, P., Jentsch, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+ion+movement+in+the+inner+ear:+insights+from+genetic+disease+and+mouse+models.">Potassium ion movement in the inner ear: insights from genetic disease and mouse models.</a> Physiology. 24, 307-316 (2009).</li><li>Chen, J., Zhao, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+an+inwardly+rectifying+K++channel+(Kir4.1)+in+the+inner+ear+and+hearing+loss.">The role of an inwardly rectifying K+ channel (Kir4.1) in the inner ear and hearing loss.</a> Neuroscience. 265, 137-146 (2014).</li><li>Steel, K. P., Barkway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+role+for+melanocytes:+their+importance+for+normal+stria+vascularis+development+in+the+mammalian+inner+ear.">Another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear.</a> Development. 107 (3), 453-463 (1989).</li><li>Ito, T., Nishio, A., Wangemann, P., Griffith, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+irreversible+hearing+loss+is+caused+by+stria+vascularis+degeneration+in+an+Slc26a4-insufficient+mouse+model+of+large+vestibular+aqueduct+syndrome.">Progressive irreversible hearing loss is caused by stria vascularis degeneration in an Slc26a4-insufficient mouse model of large vestibular aqueduct syndrome.</a> Neuroscience. 310, 188-197 (2015).</li><li>Gu, S., 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=Characterization+of+rare+spindle+and+root+cell+transcriptional+profiles+in+the+stria+vascularis+of+the+adult+mouse+cochlea.">Characterization of rare spindle and root cell transcriptional profiles in the stria vascularis of the adult mouse cochlea.</a> Scientific Reports. 10 (1), 18100 (2020).</li><li>Gow, A., 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=Deafness+in+claudin+11-null+mice+reveals+the+critical+contribution+of+basal+cell+tight+junctions+to+stria+vascularis+function.">Deafness in claudin 11-null mice reveals the critical contribution of basal cell tight junctions to stria vascularis function.</a> The Journal of Neuroscience. 24 (32), 7051-7062 (2004).</li><li>Chang, Q., 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=Virally+mediated+Kcnq1+gene+replacement+therapy+in+the+immature+scala+media+restores+hearing+in+a+mouse+model+of+human+Jervell+and+Lange-Nielsen+deafness+syndrome.">Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.</a> EMBO Molecular Medicine. 7 (8), 1077-1086 (2015).</li><li>Faridi, 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=Mutational+and+phenotypic+spectra+of+KCNE1+deficiency+in+Jervell+and+Lange-Nielsen+Syndrome+and+Romano-Ward+Syndrome.">Mutational and phenotypic spectra of KCNE1 deficiency in Jervell and Lange-Nielsen Syndrome and Romano-Ward Syndrome.</a> Human Mutation. 40 (2), 162-176 (2019).</li><li>Wangemann, 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=Loss+of+KCNJ10+protein+expression+abolishes+endocochlear+potential+and+causes+deafness+in+Pendred+syndrome+mouse+model.">Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model.</a> BMC Medicine. 2, 30 (2004).</li><li>Kitajiri, S. -. I., 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=Expression+patterns+of+claudins,+tight+junction+adhesion+molecules,+in+the+inner+ear.">Expression patterns of claudins, tight junction adhesion molecules, in the inner ear.</a> Hearing Research. 187 (1-2), 25-34 (2004).</li><li>Jagger, D. J., Nevill, G., Forge, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+properties+of+cochlear+root+cells+are+consistent+with+roles+in+potassium+recirculation+and+spatial+buffering.">The membrane properties of cochlear root cells are consistent with roles in potassium recirculation and spatial buffering.</a> Journal of the Association for Research in Otolaryngology. 11 (3), 435-448 (2010).</li><li>Ito, T., Kurata, N., Fukunaga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages+in+the+stria+vascularis.">Tissue-resident macrophages in the stria vascularis.</a> Frontiers in Neurology. 13, 818395 (2022).</li><li>Zhang, 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=VEGFA165+gene+therapy+ameliorates+blood-labyrinth+barrier+breakdown+and+hearing+loss.">VEGFA165 gene therapy ameliorates blood-labyrinth barrier breakdown and hearing loss.</a> JCI Insight. 6 (8), e143285 (2021).</li><li>Shi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+the+cochlear+intrastrial+fluid-blood+barrier+(review).">Pathophysiology of the cochlear intrastrial fluid-blood barrier (review).</a> Hearing Research. 338, 52-63 (2016).</li><li>Gu, S., 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=Identification+of+potential+Meniere's+disease+targets+in+the+adult+stria+vascularis.">Identification of potential Meniere's disease targets in the adult stria vascularis.</a> Frontiers in Neurology. 12, 630561 (2021).</li><li>Fischer, J., Ayers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleus+RNA-sequencing:+how+it's+done,+applications+and+limitations.">Single nucleus RNA-sequencing: how it's done, applications and limitations.</a> Emerging Topics in Life Sciences. 5 (5), 687-690 (2021).</li><li>Korrapati, S., 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=Single+cell+and+single+nucleus+RNA-Seq+reveal+cellular+heterogeneity+and+homeostatic+regulatory+networks+in+adult+mouse+stria+vascularis.">Single cell and single nucleus RNA-Seq reveal cellular heterogeneity and homeostatic regulatory networks in adult mouse stria vascularis.</a> Frontiers in Molecular Neuroscience. 12, 316 (2019).</li><li>Pyle, M. P., Hoa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+single-cell+sequencing+for+the+field+of+otolaryngology:+A+contemporary+review.">Applications of single-cell sequencing for the field of otolaryngology: A contemporary review.</a> Laryngoscope Investigative Otolaryngology. 5 (3), 404-431 (2020).</li><li>Hwang, B., Lee, J. H., Bang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+technologies+and+bioinformatics+pipelines.">Single-cell RNA sequencing technologies and bioinformatics pipelines.</a> Experimental &amp; Molecular Medicine. 50 (8), 1-14 (2018).</li><li>Shafer, M. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-species+analysis+of+single-cell+transcriptomic+data.">Cross-species analysis of single-cell transcriptomic data.</a> Frontiers in Cell and Developmental Biology. 7, 175 (2019).</li><li>Chen, G., Ning, B., Shi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA-Seq+technologies+and+related+computational+data+analysis.">Single-cell RNA-Seq technologies and related computational data analysis.</a> Frontiers in Genetics. 10, 317 (2019).</li><li>Longo, S. K., Guo, M. G., Ji, A. L., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+single-cell+and+spatial+transcriptomics+to+elucidate+intercellular+tissue+dynamics.">Integrating single-cell and spatial transcriptomics to elucidate intercellular tissue dynamics.</a> Nature Reviews Genetics. 22 (10), 627-644 (2021).</li><li>Kim, N., Kang, H., Jo, A., Yoo, S. A., Lee, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+single-nucleus+RNA+sequencing+in+different+cell+types+and+tissues.">Perspectives on single-nucleus RNA sequencing in different cell types and tissues.</a> Journal of Pathology and Translational Medicine. 57 (1), 52-59 (2023).</li><li>Grindberg, R. V., 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=RNA-sequencing+from+single+nuclei.">RNA-sequencing from single nuclei.</a> Proceedings of the National Academy of Sciences. 110 (49), 19802-19807 (2013).</li><li>Montgomery, S. C., Cox, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+dissection+and+immunofluorescence+of+the+adult+mouse+cochlea.">Whole mount dissection and immunofluorescence of the adult mouse cochlea.</a> Journal of Visuazlied Experiments. (107), e53561 (2016).</li></ol>

Prior to the advent of single-cell sequencing, many researchers used bulk tissue analysis, which only made it possible to analyze transcriptomes averaged across cells. In particular, single-cell and sNuc-Seq made it possible to isolate the transcriptome of a single cell or single nucleus, respectively32. In this instance, single-nucleus transcriptomes can be identified for marginal, intermediate, and basal cells, as well as spindle cells30. This enables the investigation of...

The cochlea consists of three fluid filled chambers, the scala vestibuli, scala media, and scala tympani. The scala vestibuli and scala tympani each contain perilymph, which has a high concentration of sodium (138 mM) and a low concentration of potassium (6.8 mM)1. The scala media contains endolymph, which has a high concentration of potassium (154 mM) and a low concentration of sodium (0.91 mM)1,2,3. This difference in ion concentration can be referred to as the endocochlear potential (EP), and is primarily generated by the movement of potassium ions through various ion channels and gap junctions in the stria vascularis (SV) along the lateral wall of the cochlea4,5,6,7,8,9,10,11. The SV is a heterogenous, highly vascularized tissue that lines the medial aspect of the lateral wall of the cochlea and contains three main cell types: marginal, intermediate, and basal cells12 (Figure 1).
Marginal cells are connected by tight junctions to form the most medial surface of the SV. The apical membrane faces the endolymph of the scala media and contributes to potassium ion transport into the endolymph using various channels, including KCNE1/KCNQ1, SLC12A2, and Na+-K+-ATPase (NKA)5,10,13,14. Intermediate cells are pigmented cells that reside between marginal and basal cells and facilitate potassium transport through the SV using KCNJ10 (Kir 4.1)15,16. Basal cells lie in close proximity to the lateral wall of the cochlea and are closely associated with fibrocytes of the spiral ligament to promote potassium recycling from the perilymph12. Pathology of the SV has been implicated in numerous otologic disorders17,18. Mutations in genes expressed in the major SV cell types, such as Kcnq1, Kcne1, Kcnj10, and Cldn11, can cause deafness and SV dysfunction, including the loss of EP19,20,21,22,23. In addition to the three major cell types, there are other less-studied cell types in the SV, such as spindle cells22, root cells12,24, macrophages25, pericytes26, and endothelial cells27, that have incompletely defined roles involving ionic homeostasis and the generation of EP28.
In comparison to bulk RNA-sequencing, single-nuclei RNA-sequencing (sNuc-Seq) provides information about cell heterogeneity, rather than an average of mRNA across a group of cells29, and can be particularly useful when studying the heterogenous SV30. For example, sNuc-Seq has produced transcriptional analysis that suggests there may be a role for spindle and root cells in EP generation, hearing loss, and Meniere&#39;s disease18. Further transcriptional characterization of the various SV cell types can provide us with invaluable information on the pathophysiology underlying different mechanisms and subtypes of SV-related hearing fluctuation and hearing loss. The harvest of these delicate inner ear structures is of paramount importance to optimal tissue analysis.
In this study, the microdissection approach to access and isolate the stria vascularis from the adult mouse cochlea for sNuc-Seq or immunostaining is described. Dissection of the adult mouse SV is required to understand various SV cell types and further characterize their role in hearing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >10-&#181;m filter (Polyethylenterephthalat)</td>
			<td >PluriSelect</td>
			<td >#43-50010-01</td>
			<td >Filter tissue during sNuc-Seq</td>
		</tr>
		<tr >
			<td >18 x 18 mm cover glass</td>
			<td >Fisher Scientific</td>
			<td >12-541A</td>
			<td >Cover slip to mount SV</td>
		</tr>
		<tr >
			<td >30-&#181;m filter (Polyethylenterephthalat)</td>
			<td >PluriSelect</td>
			<td >#43-50030-03</td>
			<td >Filter tissue during sNuc-Seq</td>
		</tr>
		<tr >
			<td >75 x 25 mm Superfrost Plus/Colorforst Plus Microslide</td>
			<td >Daigger</td>
			<td >EF15978Z</td>
			<td >Microslide to mount SV on</td>
		</tr>
		<tr >
			<td >C57BL/6J Mice</td>
			<td >The Jackson Laboratory</td>
			<td >RRID: IMSR_JAX:000664</td>
			<td >General purpose mouse strain that has pigment more easily seen in the intermediate cells of the SV.</td>
		</tr>
		<tr >
			<td >Cell Counter</td>
			<td >Logos Biosystems</td>
			<td >L20001</td>
			<td >Used for cell counting</td>
		</tr>
		<tr >
			<td >Chalizon curette 5&#39;&#39;, size 3 2.5 mm</td>
			<td >Biomedical Research Instruments</td>
			<td >15-1020</td>
			<td >Used to transfer SV</td>
		</tr>
		<tr >
			<td >Chromium Next GEM single Cell 3&#39; GEM Kit v3.1</td>
			<td >Chromium</td>
			<td >PN-1000141</td>
			<td >Generates single cell 3&#39; gene expression libraries</td>
		</tr>
		<tr >
			<td >Clear nail polish</td>
			<td >Fisher Scientific</td>
			<td >NC1849418</td>
			<td >Used for sealing SV mount</td>
		</tr>
		<tr >
			<td >Corning Falcon Standard Tissue Culture Dishes, 24 well</td>
			<td >Corning</td>
			<td >08-772B</td>
			<td >Culture dish used to hold specimen during dissection</td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Invitrogen</td>
			<td >D1306, RRID: AB_2629482</td>
			<td >Stain used for nucleus labeling</td>
		</tr>
		<tr >
			<td >Dounce homogenizer</td>
			<td >Sigma-Aldrich</td>
			<td >D8938</td>
			<td >Used to homogenize tissue for sNuc-seq</td>
		</tr>
		<tr >
			<td >Dumont #5 Forceps</td>
			<td >Fine Science Tools</td>
			<td >11252-30</td>
			<td >General forceps for dissection</td>
		</tr>
		<tr >
			<td >Dumont #55 Forceps</td>
			<td >Fine Science Tools</td>
			<td >11255-20</td>
			<td >Forceps with fine tip that makes SV manipulation easier</td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ThermoFisher</td>
			<td >16000044</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Glue stick</td>
			<td >Fisher Scientific</td>
			<td >NC0691392</td>
			<td >Used for mounting SV</td>
		</tr>
		<tr >
			<td >GS-IB4 Antibody</td>
			<td >Molecular Probes</td>
			<td >I21411, RRID: AB-2314662</td>
			<td >Antibody used for capillary labeling</td>
		</tr>
		<tr >
			<td >KCNJ10-ZsGreen Mice</td>
			<td >n/a</td>
			<td >n/a</td>
			<td >Transgenic mouse that expresses KCNJ10-ZsGreen, partiularly in the intermediate cells of the SV.</td>
		</tr>
		<tr >
			<td >MgCl2</td>
			<td >ThermoFisher</td>
			<td >AM9530G</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Mounting reagent</td>
			<td >ThermoFisher</td>
			<td >#S36940</td>
			<td >Mounting reagent for SV</td>
		</tr>
		<tr >
			<td >Multiwell 24 well plate</td>
			<td >Corning</td>
			<td >#353047</td>
			<td >Plate used for immunostaining</td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >ThermoFisher</td>
			<td >AAJ216183</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Nonidet P40</td>
			<td >Sigma-Aldrich</td>
			<td >9-16-45-9</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Nuclease free water</td>
			<td >ThermoFisher</td>
			<td >4387936</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Orbital shaker</td>
			<td >Silent Shake</td>
			<td >SYC-2102A</td>
			<td >Used for steps of immunostaining</td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >ThermoFisher</td>
			<td >J61196.AP</td>
			<td >Used for steps of immunostaining and dissection</td>
		</tr>
		<tr >
			<td >RNA Later</td>
			<td >Invitrogen</td>
			<td >AM7021</td>
			<td >Used for preservation of SV for sNuc-Seq</td>
		</tr>
		<tr >
			<td >Scizzors</td>
			<td >Fine Science Tools</td>
			<td >14058-09</td>
			<td >Used for splitting mouse skull</td>
		</tr>
		<tr >
			<td >Tris-HCl</td>
			<td >Sigma-Aldrich</td>
			<td >15506017</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Trypan blue stain</td>
			<td >Gibco</td>
			<td >15250061</td>
			<td >Used for cell counting</td>
		</tr>
		<tr >
			<td >Tween20</td>
			<td >ThermoFisher</td>
			<td >AAJ20605AP&#160;</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Zeiss STEMI SV 11 Apo stereomicroscope</td>
			<td >Zeiss</td>
			<td >n/a</td>
			<td >Microscope used for dissections</td>
		</tr>
	</tbody>
</table>

dissection of adult mouse stria vascularis for single-nucleus sequencing or immunostaining

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. 
Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

We present a method to isolate the SV to be used for either sNuc-Seq or immunostaining. The relevant anatomy (Figure 1) of the cochlea relative to the SV can help users better understand the organization of the SV and steps of the dissection protocol.
Each step of this microdissection of SV from a P30 mouse is detailed in the associated video, and snapshots of the key steps of this dissection and isolation of SV are presented in Figure 2</stro...

Watch this Scientific Journal Video about Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining at JoVE.com

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining (Video) | JoVE 

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.