Name: three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary
Uploaded: 2017-12-10T14:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary at JoVE.com

This study was supported by grants from the Chinese Special Fund for Postdocs (No. 2014T70392 to YF), the National Natural Science Foundation of China (No. 81673766 to YF), the New Teacher Priming Fund, the Zuoxue Foundation of Fudan University, and the Development Project of Shanghai Peak Disciplines-Integrative Medicine (20150407).

All procedures involving animal subjects followed the guidelines of the Animal Ethics Committee at Shanghai Medical College, Fudan University (Approval number 20160225-013).
1. Preparation of the Transparent Mouse Ovary
<ol>
	<li>Preparation of the solutions
	<ol>
		<li>Prepare phosphate-buffered saline (PBS) solution (1 M, pH 7.6) with 0.1% Triton X-100 (PBST). To make 1 L of 10x PBS stock solution, mix 87 g of NaCl, 3.1 g of NaH2PO4 &#183; 2H2</s...

<ol>
	<li>Brown, H. M., Russell, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+and+lymphatic+vasculature+in+the+ovary:+development,+function+and+disease.">Blood and lymphatic vasculature in the ovary: development, function and disease.</a> Hum Reprod Update. 20 (1), 29-39 (2014).</li><li>McFee, R. 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=Inhibition+of+vascular+endothelial+growth+factor+receptor+signal+transduction+blocks+follicle+progression+but+does+not+necessarily+disrupt+vascular+development+in+perinatal+rat+ovaries.">Inhibition of vascular endothelial growth factor receptor signal transduction blocks follicle progression but does not necessarily disrupt vascular development in perinatal rat ovaries.</a> Biol Reprod. 81 (5), 966-977 (2009).</li><li>Coveney, D., Cool, J., Oliver, T., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-dimensional+analysis+of+vascularization+during+primary+development+of+an+organ,+the+gonad.">Four-dimensional analysis of vascularization during primary development of an organ, the gonad.</a> Proc Natl Acad Sci U S A. 105 (20), 7212-7217 (2008).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9 (7), 1682-1697 (2014).</li><li>Schindelin, 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=Fiji:+an+Open+Source+platform+for+biological+image+analysis.">Fiji: an Open Source platform for biological image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Cao, G., Fehrenbach, M. L., Williams, J. T., Finklestein, J. M., Zhu, J. X., Delisser, 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=Angiogenesis+in+platelet+endothelial+cell+adhesion+molecule-1-null+mice.">Angiogenesis in platelet endothelial cell adhesion molecule-1-null mice.</a> Am J Pathol. 175 (2), 903-915 (2009).</li><li>Manni, L., Holm&#228;ng, A., Lundeberg, T., Aloe, L., Stener-Victorin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+expression+of+alpha+(1)-+and+beta+(2)-adrenoceptors+and+p75+neurotrophin+receptors+in+rats+with+steroid-induced+polycystic+ovaries.">Ovarian expression of alpha (1)- and beta (2)-adrenoceptors and p75 neurotrophin receptors in rats with steroid-induced polycystic ovaries.</a> Auton Neurosci. 118 (1 - 2), 79-87 (2005).</li><li>Chourasia, T. K., Chaube, R., Singh, V., Joy, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annual+and+periovulatory+changes+in+tyrosine+hydroxylase+activity+in+the+ovary+of+the+catfish+Heteropneustes+fossilis.">Annual and periovulatory changes in tyrosine hydroxylase activity in the ovary of the catfish Heteropneustes fossilis.</a> Gen Comp Endocrinol. 166 (1), 111-116 (2010).</li><li>Feng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+reveals+dynamics+of+ovarian+follicular+architecture+and+vasculature+in+three-dimensions.">CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.</a> Sci Rep. 7, 44810 (2017).</li><li>Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T., Ueda, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+principles+in+tissue+clearing+and+staining+protocols+for+whole-body+cell+profiling.">Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.</a> Annu Rev Cell Dev Biol. 32, 713-741 (2016).</li><li>Liang, H., Schofield, E., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Serotonergic+Fibers+in+the+Mouse+Spinal+Cord+Using+the+CLARITY/CUBIC+Technique.">Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique.</a> J Vis Exp. (108), e53673 (2016).</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 (4), 945-958 (2014).</li><li>Phillips, J., Laude, A., Lightowlers, R., Morris, C. M., Turnbull, D. M., Lax, N. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+passive+CLARITY+and+immunofluorescent+labelling+of+multiple+proteins+in+human+cerebellum:+understanding+mechanisms+of+neurodegeneration+in+mitochondrial+disease.">Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease.</a> Sci Rep. 6, 26013 (2016).</li><li>Roberts, D. G., Johnsonbaugh, H. B., Spence, R. D., MacKenzie-Graham, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+clearing+of+the+mouse+central+nervous+system+using+passive+CLARITY.">Optical clearing of the mouse central nervous system using passive CLARITY.</a> J Vis Exp. (112), (2016).</li><li>Woo, J., Lee, M., Seo, J. M., Park, H. S., Cho, Y. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+optical+transparency+of+rodent+tissues+by+modified+PACT-based+passive+clearing.">Optimization of the optical transparency of rodent tissues by modified PACT-based passive clearing.</a> Exp Mol Med. 48 (12), e274 (2016).</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 (7449), 332-337 (2013).</li><li>Chung, A. S., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+pathological+angiogenesis.">Developmental and pathological angiogenesis.</a> Annu Rev Cell Dev Biol. 27, 563-584 (2011).</li><li>Rodgers, R. J., Irving-Rodgers, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+ovarian+follicular+antrum+and+follicular+fluid.">Formation of the ovarian follicular antrum and follicular fluid.</a> Biol Reprod. 82 (6), 1021-1029 (2010).</li><li>Siu, M. K. Y., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-follicle+barrier+(BFB)+in+disease+and+in+ovarian+function.">The blood-follicle barrier (BFB) in disease and in ovarian function.</a> Adv Exp Med Biol. 763, 186-192 (2014).</li><li>Dodt, H. U., 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=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat Methods. 4, 331-336 (2007).</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>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>Kuwajima, 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=Clear(T):+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">Clear(T): a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140, 1364-1368 (2013).</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>Lai, H. 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=Rationalisation+and+validation+of+an+acrylamide-free+procedure+in+three-dimensional+histological+imaging.">Rationalisation and validation of an acrylamide-free procedure in three-dimensional histological imaging.</a> PLOS ONE. 11, e0158628 (2016).</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>Tainaka, 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=Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization.">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159, 911-924 (2014).</li><li>Liu, A. K. L., Lai, H. M., Chang, R. C. C., Gentleman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-of-acrylamide+SDS-based+tissue+clearing+(FASTClear):+A+novel+protocol+of+tissue+clearing+for+three-dimensional+visualisation+of+human+brain+tissues.">Free-of-acrylamide SDS-based tissue clearing (FASTClear): A novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.</a> Neuropathol Appl Neurobiol. 43, 346-351 (2016).</li><li>Xu, 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=Fast+free-of-acrylamide+clearing+tissue+(FACT)-an+optimized+new+protocol+for+rapid,+high-resolution+imaging+of+three-dimensional+brain+tissue.">Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of three-dimensional brain tissue.</a> Sci Rep. 7, 9895 (2017).</li><li>Migone, F. F., Cowan, R. G., Williams, R. M., Gorse, K. J., Zipfel, W. R., Quirk, S. M. <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+reveals+an+essential+role+of+vasoconstriction+in+rupture+of+the+ovarian+follicle+at+ovulation.">In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.</a> Proc Natl Acad Sci U S A. 113, 2294-2299 (2016).</li><li>Malki, S., Tharp, M. E., Bortvin, 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+whole-mount+approach+for+accurate+quantitative+and+spatial+assessment+of+fetal+oocyte+dynamics+in+mice.">A whole-mount approach for accurate quantitative and spatial assessment of fetal oocyte dynamics in mice.</a> Biol Reprod. 93 (113), (2015).</li></ol>

In the current study, we present 3D imaging to evaluate the relationships between capillaries and individual growing follicles. In our previous work using the same protocol 9, we studied the roles of large vasculature, interactions between follicles, and the location of follicles in intact mouse ovaries. The passive CLARITY approach allowed us to study micro- and macro-vasculatures, folliculogenesis, and the interrelationships between the corpora lutea and follicles as well as to reconstruct the o...

The follicle is the fundamental structural and functional unit of the ovary, and its development is highly related to the vasculature within the ovary. Blood vessels supply nutrition and hormones to the follicles and thus play important roles in the growth and maturation of follicles1.
A combination of technologies, including selective blood vessel markers, transgenic mouse models, and pharmaceutical development, have increased our knowledge about ovarian vascular networks, angiogenesis, and the function of blood vessels in folliculogenesis. The ovary is known as an active organ because it remodels various tissues and vascular networks during folliculogenesis and ovulation. Such active remodeling in the size and structure of vessels is required for the biological function of developing and recruiting follicles.
Traditional histological and histomorphometric methods using ovarian sections and immunolabeling of blood vessels are limited to two-dimensional (2D) images2. With the development of three-dimensional (3D) reconstruction technologies, 2D images of tissue slices can be overlapped to make a 3D structure, but this method still has some limitations &#8212; sectioning of the tissue can destroy the microstructures, some parts of the tissue are often missing, and significant labor is involved in making 3D reconstructions from images obtained from slices. Whole-tissue 3D imaging with confocal microscopy can overcome many of these limitations, but these methods are limited to the evaluation of angiogenesis in the embryonic ovary3. Using whole tissue clearing methods such as CLARITY4 can increase the visualized volume so as to solve these problems in postnatal and adult ovaries, and such methods provide optical clearance of the ovary without any structural deformations. Imaging of the 3D architecture of the intact ovary provides an accurate image database for image analysis software, such as the Imaris software package used in this work.
Remodeling of the ovary throughout adulthood is part of a dynamic physiological system, and this makes the ovary an excellent model for investigations into the regulation of angiogenesis. Furthermore, evaluating the role of ovarian blood vessels in pathologic conditions of the female reproductive system such as polycystic ovary syndrome or ovarian cancers can be studied through whole ovarian tissue imaging. The development of the passive CLARITY method and the use of advanced image analysis software have provided detailed spatial information on the relationships between blood vessels and ovarian structures such as follicles.

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		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Acrylamide</td>
			<td style="width:189px;">Vetec</td>
			<td style="width:127px;">v900845</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/vetec/v900845</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 488 (Dilution 1:50)&#160;</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11039</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 594 (Dilution 1:50)</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11012</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Bisacrylamide</td>
			<td style="width:189px;">Amresco</td>
			<td style="width:127px;">172</td>
			<td style="width:861px;">http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Black wall glass bottom dish (Willco-Dish)</td>
			<td style="width:189px;">Ted Pella</td>
			<td style="width:127px;">14032</td>
			<td style="width:861px;">http://www.tedpella.com/section_html/706dish.htm#black_wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Boric acid</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10004818</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10004818</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Disodium hydrogen phosphate dodecahydrate (Na2HPO4 12H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10020318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10020318</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FocusClear</td>
			<td style="width:189px;">Celexplorer</td>
			<td style="width:127px;">FC-102</td>
			<td style="width:861px;">http://www.celexplorer.com/product_list.asp?MainType=107&#38;BRDarea=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Parafilm</td>
			<td style="width:189px;">Bemis</td>
			<td style="width:127px;">PM996</td>
			<td style="width:861px;">http://www.parafilm.com/products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Paraformaldehyde</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">80096618</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=80096618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab28364</td>
			<td style="width:861px;">http://www.abcam.com/cd31-antibody-ab28364.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Photoinitiator VA044</td>
			<td style="width:189px;">Wako</td>
			<td style="width:127px;">va-044/225-02111</td>
			<td style="width:861px;">http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium azide</td>
			<td style="width:189px;">Sigma</td>
			<td style="width:127px;">S2002</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium chloride (NaCl)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019318</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Sodium dihydrogen phosphate dihydrate (NaH2PO4 2H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">20040718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=20040718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium dodecyl sulfate</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30166428</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30166428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium hydroxide (NaOH)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Triton X-100</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30188928</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30188928</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Tyrosine hydroxylase (TH, Dilution 1:50)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab76442</td>
			<td style="width:861px;">http://www.abcam.com/tyrosine-hydroxylase-phospho-s40-antibody-ab51206.html</td>
		</tr>
	</tbody>
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three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

We adapted the passive CLARITY method into a quick and simple method for passive ovary clearing while preserving the follicular and vascular architecture and obtaining the highest fluorescent signal from labeled markers of vessels and follicles. The 3D architecture of the follicular vasculature was determined by immunostaining for CD31, a marker for endothelial cells6. CD31 staining in the ovaries of adult mice was traced using the Filament algorithm and showed int...

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Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Research

JoVE Journal

Biology

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