Name: imaging cleared embryonic and postnatal hearts at single-cell resolution
Uploaded: 2016-10-07T13:00:00
Description: Watch this Scientific Journal Video about Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution at JoVE.com

We thank M.W. laboratory members for scientific discussion. This work is supported by AHA [13SDG16920099] to M.W., and by National Heart, Lung, and Blood Institute grants [R01HL121700] to M.W. Images were captured in the Imaging Core Facility at the Albany Medical College.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Albany Medical College and performed according to the NIH Guide for the Care and Use of Laboratory Animals.
1. Solution Preparations
NOTE: The Rosa26CreERT2 15, R26R-Confetti16, &#945;MHC-Cre17,and Rosa26-mTmG (mTmG)20 mouse lines were purchased commercially. cTnT-Cre18 ...

<ol>
	<li>Vincent, S. D., Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+heart:+the+origin+and+regulation+of+cardiac+progenitor+cells.">How to make a heart: the origin and regulation of cardiac progenitor cells.</a> Curr Top Dev Biol. 90, 1-41 (2010).</li><li>Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+discoveries+in+cardiac+biology.">A decade of discoveries in cardiac biology.</a> Nat Med. 10, 467-474 (2004).</li><li>Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+in+the+evolution+and+development+of+the+heart.">Gene regulatory networks in the evolution and development of the heart.</a> Science. 313, 1922-1927 (2006).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+congenital+heart+disease.">The developmental genetics of congenital heart disease.</a> Nature. 451, 943-948 (2008).</li><li>Mohun, T. J., Weninger, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embedding+embryos+for+high-resolution+episcopic+microscopy+(HREM).">Embedding embryos for high-resolution episcopic microscopy (HREM).</a> Cold Spring Harb Protoc. , 678-680 (2012).</li><li>Soufan, A. 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=Three-dimensional+reconstruction+of+gene+expression+patterns+during+cardiac+development.">Three-dimensional reconstruction of gene expression patterns during cardiac development.</a> Physiol Genomics. 13, 187-195 (2003).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162, 246-257 (2015).</li><li>Susaki, E. 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=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</li><li>Becker, K., Jahrling, N., Saghafi, S., Weiler, R., Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+clearing+and+dehydration+of+GFP+expressing+mouse+brains.">Chemical clearing and dehydration of GFP expressing mouse brains.</a> PLoS One. 7, e33916 (2012).</li><li>Erturk, 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=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat Protoc. 7, 1983-1995 (2012).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16, 1154-1161 (2013).</li><li>Chung, K., 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497, 332-337 (2013).</li><li>Yang, B., 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+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158, 945-958 (2014).</li><li>Hama, 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=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Badea, T. C., Wang, Y., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+noninvasive+genetic/pharmacologic+strategy+for+visualizing+cell+morphology+and+clonal+relationships+in+the+mouse.">A noninvasive genetic/pharmacologic strategy for visualizing cell morphology and clonal relationships in the mouse.</a> J Neurosci. 23, 2314-2322 (2003).</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>Agah, 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=Gene+recombination+in+postmitotic+cells.+Targeted+expression+of+Cre+recombinase+provokes+cardiac-restricted,+site-specific+rearrangement+in+adult+ventricular+muscle+in+vivo.">Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo.</a> J Clin Invest. 100, 169-179 (1997).</li><li>Jiao, K., 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=An+essential+role+of+Bmp4+in+the+atrioventricular+septation+of+the+mouse+heart.">An essential role of Bmp4 in the atrioventricular septation of the mouse heart.</a> Genes Dev. 17, 2362-2367 (2003).</li><li>Wu, B., 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=Endocardial+cells+form+the+coronary+arteries+by+angiogenesis+through+myocardial-endocardial+VEGF+signaling.">Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling.</a> Cell. 151, 1083-1096 (2012).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li><li>Wu, M., 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=Epicardial+spindle+orientation+controls+cell+entry+into+the+myocardium.">Epicardial spindle orientation controls cell entry into the myocardium.</a> Dev Cell. 19, 114-125 (2010).</li><li>Zhao, C., 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=Numb+family+proteins+are+essential+for+cardiac+morphogenesis+and+progenitor+differentiation.">Numb family proteins are essential for cardiac morphogenesis and progenitor differentiation.</a> Development. 141, 281-295 (2014).</li><li>Kaufman, M. H., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The atlas of mouse development. , (1992).</li><li>Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo : a laboratory manual. , (2003).</li><li>Buckingham, M., Meilhac, S., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+mammalian+heart+from+two+sources+of+myocardial+cells.">Building the mammalian heart from two sources of myocardial cells.</a> Nat Rev Genet. 6, 826-835 (2005).</li><li>Kelly, R. G., Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anterior+heart-forming+field:+voyage+to+the+arterial+pole+of+the+heart.">The anterior heart-forming field: voyage to the arterial pole of the heart.</a> Trends Genet. 18, 210-216 (2002).</li><li>Verzi, M. P., McCulley, D. J., De Val, S., Dodou, E., Black, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+right+ventricle,+outflow+tract,+and+ventricular+septum+comprise+a+restricted+expression+domain+within+the+secondary/anterior+heart+field.">The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field.</a> Dev Biol. 287, 134-145 (2005).</li><li>Ward, C., Stadt, H., Hutson, M., Kirby, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+the+secondary+heart+field+leads+to+tetralogy+of+Fallot+and+pulmonary+atresia.">Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia.</a> Dev Biol. 284, 72-83 (2005).</li><li>Echelard, Y., Vassileva, G., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis-acting+regulatory+sequences+governing+Wnt-1+expression+in+the+developing+mouse+CNS.">Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS.</a> Development. 120, 2213-2224 (1994).</li><li>Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P., Sucov, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+the+mammalian+cardiac+neural.">Fate of the mammalian cardiac neural.</a> Developement. 127, 1607-1616 (2000).</li><li>Viragh, S., Challice, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+the+epicardium+and+the+embryonic+myocardial+circulation+in+the+mouse.">The origin of the epicardium and the embryonic myocardial circulation in the mouse.</a> Anat Rec. 201, 157-168 (1981).</li><li>Wu, M., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numb+family+proteins:+novel+players+in+cardiac+morphogenesis+and+cardiac+progenitor+cell+differentiation.">Numb family proteins: novel players in cardiac morphogenesis and cardiac progenitor cell differentiation.</a> Biomolecular concepts. 6, 137-148 (2015).</li><li>Li, 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=Single-Cell+Lineage+Tracing+Reveals+that+Oriented+Cell+Division+Contributes+to+Trabecular+Morphogenesis+and+Regional+Specification.">Single-Cell Lineage Tracing Reveals that Oriented Cell Division Contributes to Trabecular Morphogenesis and Regional Specification.</a> Cell reports. 15, 158-170 (2016).</li><li>Alkaabi, K. M., Yafea, A., Ashraf, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+on+thermal-+and+chemical-induced+denaturation+of+GFP.">Effect of pH on thermal- and chemical-induced denaturation of GFP.</a> Appl Biochem Biotechnol. 126, 149-156 (2005).</li><li>Captur, 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=Morphogenesis+of+myocardial+trabeculae+in+the+mouse+embryo.">Morphogenesis of myocardial trabeculae in the mouse embryo.</a> J Anat. , (2016).</li></ol>

The embryo isolation is a very critical step. E9.5 embryos are very fragile and small in size, so extra care should be taken not to damage the embryo/heart structures during isolation. The non-embryonic extra layers enveloping the embryo/heart should be removed carefully especially when imaging the whole embryo. This allows antibody and clearing mixture penetration deep inside the embryonic tissues, and also helps in removing the background signal when imaging. Multiple antibodies including antibodies against PECAM, acet...

Cardiac morphogenesis is a sequential event that requires the spatiotemporal organization of four different types of cardiac progenitor cells into distinct sectors of the heart, and also requires multiple genetic regulatory networks to orchestrate this process to form the functional heart1,2. Cardiac specification, differentiation, patterning, and chamber maturation are regulated by cardiogenic transcription factors3. Genetic mutation or posttranscriptional aberration of these factors in cardiac progenitor cells could result in either embryonic lethality or congenital heart defects (CHD)4. The study of cardiac morphogenesis requires an understanding of inherent structural details in three dimensions (3D) and single labeled cardiac progenitor cell lineage tracing during cardiac development will promote the understanding of cardiac morphogenesis. A number of high-resolution section based tomography methods have been developed in the past few decades to image organ structure5,6; however, these methods require expensive, specialized instruments, extensive labor, and lack detailed structural organization at single cell resolution in the final volumetric reconstructed image7,8.
3D volumetric imaging at the single cell level provides a means to study progenitor cell differentiation and cellular behavior in vivo7. However, tissue light scattering remains the primary obstacle to imaging cells and structures in 3D deep inside the intact heart. Lipids are a major source of light scattering, and the removal of lipids and/or adjustment of the refractive index difference between lipids and their surrounding areas are potential approaches for increasing tissue transparency8. In the past several years, a number of tissue clearing methods were developed, which reduce tissue opacity and light scattering, like BABB (benzyl alcohol and benzyl benzoate mixture) and DBE (tetrahydrofuran and dibenzylether); but in these methods, fluorescence quenching remains an issue8-10. The solvent based hydrophilic methods, such as SeeDB (fructose/thioglycerol) and 3DISCO (dichloromethane/dibenzylether), preserve fluorescent signals, but do not render the whole organ transparent7,8,11. In comparison, the CLARITY tissue-clearing method renders the organ transparent, but it requires a specialized electrophoresis device to remove lipids8,12, as does PACT (passive clarity technique), which also requires hydrogel embedding7,13. For detailed information regarding all available tissue-clearing methods, refer to Table 1 in Richardson and Lichtman, et al.7.
In 2011, Hama et al. serendipitously discovered a hydrophilic mixture &#39;Scale&#39; (urea, glycerol and Triton X-100 mixture) that renders the mouse brain and embryo transparent while completely preserving fluorescent signals from labeled clones14. This allows for the imaging of the intact brain at a depth of several millimeters and large-scale reconstruction of neuronal populations and projections at a subcellular resolution. Susaki et al. further improved Scale by adding aminoalcohols and developed the &#39;CUBIC&#39; (clear, unobstructed brain imaging cocktails and computational analysis) tissue clearing method, which increased phospholipid solubilization, reduced clearing time, and allowed for multicolor fluorescent imaging8. In the present study, taking advantage of the Scale and CUBIC tissue clearing techniques and high resolution 3D optical sectioning, individual clones inside the heart during cardiogenesis were traced using Rosa26CreERT2 15, R26R-Confetti16, &#945;MHC-Cre17, cTnT-Cre18, Nfatc1-Cre19, and Rosa26-mTmG (mTmG)20 mouse lines. The combination of the whole mount staining (WMS) method developed previously21,22 with tissue clearing methods further allowed for the staining of other proteins in labeled clones and for the study of their behavior in a 3D volumetric context. The combination of tissue clearing and WMS allows for a better understanding of the roles of different genes and proteins during cardiac development, and the etiology of congenital heart defects. This protocol can be applied to study other progenitor cell differentiation, cellular behavior, and organ morphogenesis events during development.

<table border="0" cellpadding="0" cellspacing="0" width="950">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">2,2&#8242;,2&#8242;&#8217;-nitrilotriethanol&#160;</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">90279</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% Paraformaldehyde in PBS</td>
			<td style="width:192px;">Affymetrix</td>
			<td>19943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td style="width:192px;">Fischer Scientific&#160;</td>
			<td style="width:211px;">BP16000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N,N&#8217;,N&#8217;-tetrakis(2-hydroxypropyl) ethylenediamine&#160;</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">122262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">P5368-10PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton&#160;X-100</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">U-1250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Sucrose</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">84097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Glycerol</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">G8773</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Tamoxifen</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">T5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Sunflower seed oil</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">S5007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">PECAM (CD31)</td>
			<td>BD Pharmingen&#160;</td>
			<td style="width:211px;">550274</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647&#160;</td>
			<td>Invitrogen</td>
			<td>A-21247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">DAPI nuclear stain</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">D9542</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">37oC Incubator</td>
			<td style="width:192px;">Thermoscientific Fischer</td>
			<td style="width:211px;">Heratherm, Compact Microbiological Incubators</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48 well plates</td>
			<td style="width:192px;">Cell Treat</td>
			<td style="width:211px;">229148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Analytical Balance</td>
			<td style="width:192px;">Metler Toledo</td>
			<td>PB153-S/FACT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510 confocal microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Disecting Microscope</td>
			<td style="width:192px;">Unitron</td>
			<td style="width:211px;">Z850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Observer. Z1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Germinator 500 Glass Bead Sterilizer</td>
			<td style="width:192px;">CellPoint Scientific</td>
			<td style="width:211px;">GER-5287-120V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Light Source</td>
			<td style="width:192px;">SCHOTT</td>
			<td style="width:211px;">ACE I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pair of Scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish 60x15mm&#160;</td>
			<td align="left">TPP Techno Plastic Products AG</td>
			<td>93060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Rocker II&#160; Platform Rocker</td>
			<td>Boekel Scientific</td>
			<td style="width:211px;">260350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scintillating tubes</td>
			<td style="width:192px;">Fischer Scientific&#160;</td>
			<td>03-337-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipette</td>
			<td style="width:192px;">Samco Scientific</td>
			<td style="width:211px;">202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tweezers</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cleared embryonic and postnatal hearts at single-cell resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

Imaging the cleared embryonic heart
Vertebrate heart formation is a spatiotemporally regulated morphogenic process and depends on the organization and differentiation of progenitor cells from four different sources1. Cells from the first heart field of the cardiac crescent will fold toward the ventral midline to form a linear heart tube. The cells from the second heart field, initially residing dorsomedially to the fi...

Watch this Scientific Journal Video about Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution at JoVE.com

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

The overall goal of this procedure is to volumetrically image fluorescent protein labeled cells deep inside intact hearts, to study gene function at single cell-resolution during cardiac development and in postnatal hearts. This method can help to answer key questions during cardiac development, such as the study of the gene functions and helping image at single-cell resolution during cardiac development and animidal artifacts. The main advantage of this method is it is relatively simple, inexpensive, and pro-wise in that special temporal analysis of the gene and protein functions at single-cell resolution in intact embryo or heart.Although we use this system to study heart development in mice, you can also use it to study other organ systems, such as muscles, brain, and lungs and in other model organisms as well. Fluorescent protein positive embryos from Tamoxifren-treated R-26 Cre-RET2 confetti dams are used here. Begin staining by immersing each paraformaldehyde-fixed embryo in one milliliter of 0.1%PBST in the wells of a 48 well plate.Incubate for two hours on a plate shaker at room temperature to permeabilize the tissue. Then, replace the permeabilization solution with one mL of blocking buffer and incubate, with shaking, for two hours at room temperature or overnight at four degrees celsius. Next, prepare a one to 100 dilution of the endothelial-cell specific antibody, PECAM, in 0.3 milliliters of blocking buffer for each well and add this to the wells containing the embryos.Incubate for 48 hours with shaking at four degrees celsius. Following the incubation, wash the embryos five times with PBST for 30 minutes each time to remove excess primary antibody from the tissue. Next, immerse the embryos in Alexa Fluor 647 goat antivat secondary antibody solution and incubate with shaking for 48 hours at four degrees celsius, while protected from light.48 hours later, after washing out before, post-fix the embryos with 4%PFA for one hour at room temperature with shaking. Then, after washing twice with PBS for five minutes each time, incubate the embryos with DAPI nuclear stain for ten minutes. Follow with two PBS washes of five minutes each, then proceed to tissue clearing.The tissue clearing is the most critical step in this protocol in order to image the heart at a single-cell resolution. Also, the selection of the clearing method either scale or CUBIC, is also very important in order to preserve fluorescent signals in the intact embryonic heart. Transfer the washed, stained embryos into a scintillation vial wrapped in aluminum foil, that contains one milliliter of scale A2 solution.Incubate at four degrees celsius with occasional gentle shaking by hand for 48 hours. After the scale A2 incubation, replace the solution in the vial with one milliliter of scale B4 solution, and incubate at four degrees celsius with occasional gentle shaking for another 48 hours. Incubate the embryos for another 48 hours with one milliliter of scale 70 at four degrees celsius with occasional gentle shaking as before.Lastly, immerse the scale cleared sample in one milliliter of 90%glycerol and store at four degrees celsius until imaging. To image, transfer the sample to a glass-bottom petri dish with a minimal amount of 90%glycerol and image with a confocal microscope equipped with two photon capabilities. For CUBIC clearing, immerse each embryo in one milliliter of CUBIC one with occasional gentle shaking for 48 hours at 37 degrees celsius.After 48 hours, replace the solution with the same volume of fresh CUBIC reagent one and incubate the sample for an additional 48 hours, as before. Following the incubation, wash the CUBIC one treated embryos with PBS several times with gentle shaking by hand to remove the excess CUBIC one solution. Then, immerse the embryos in CUBIC two solution for 48 hours at 37 degrees celsius with occasional gentle shaking by hand.If there will be a delay before imaging, sequentially incubate the samples with one milliliter of 30%50%and 70%glycerol and PBS for 30 minutes each. After moving through the series, transfer to 90%glycerol solution for storage at four degrees celsius. When ready, image in a glass-bottom petri dish with 90%glycerol, as before.This movie shows optical sections of a scale-treated E 8.75 heart, from heart surface to lumen. Yellow marks the PECAM expression and all the cardiomyocytes appear green. The depth is 110.4 microns and there are 93 slices in total.This is the 3D surface picture of the reconstructed heart just shown. Again, yellow marks PECAM expression, and green labels all the cardiomyocytes. This movie shows sections of a single progenitor clone from a scale-treated E 9.5 heart in the outflow tract region.Yellow marks the PECAM expression and the clone appears green. The depth is 36 microns and there are 10 slices in total. This movie shows sections of a CUBIC-treated P two heart from surface to lumen.The depth is 130 microns with three microns per section. This movie shows sections of a CUBIC-treated E 17.5 heart from surface to lumen. The depth is 630 microns with six microns per section.This is a 3D surface picture of the heart just shown, reconstructed via the 3D reconstruction software. When attempting this procedure, it is important to remember that the embryo is fragile and heart pathology can be disturbed easily. Following this procedure, other protein can be realized, using staining to study the gene function at a post-ost lation and lather.

imaging-cleared-embryonic-postnatal-hearts-at-single-cell

Research

JoVE Journal

Developmental Biology

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 &#956;m, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.