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

Deze studie werd ondersteund door subsidies van de Chinese speciale fonds voor Postdocs (nr. 2014T70392 aan de YF), de nationale Natural Science Foundation van China (nr. 81673766 aan de YF), het nieuwe leraar Priming Fonds, de oprichting van de Zuoxue van Fudan Universiteit en de ontwikkeling Project van Shanghai piek Disciplines-integratieve geneeskunde (20150407).

Alle procedures waarbij dierlijke onderwerpen volgde de richtsnoeren van de ethische commissie van het dier aan Shanghai Medical College, Fudan Universiteit (erkenningsnummer 20160225-013).
1. bereiding van de transparante muis eierstok
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Bereiding van de oplossingen
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<li>Bereid met fosfaat gebufferde zoutoplossing (PBS)-oplossing (1 M, pH 7,6) met 0,1% Triton X-100 (PBST). Om 1 L van 10 x PBS stockoplossing, meng 87 g NaCl, 3.1 g van NaH2PO4</sub...

<ol>
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In de huidige studie presenteren we 3D-imaging om te evalueren van de relaties tussen de haarvaten en individuele groeiende follikels. In onze vorige werk met behulp van de dezelfde protocol 9, studeerde we de rol van grote therapieën, interacties tussen follikels en de locatie van de follikels in de eierstokken intact muis. De passieve houding van de duidelijkheid ons toegestaan om te studeren van micro - en macro-vasculatures, folliculogenese en de relaties tussen de corpora lutea en de follike...

De follikel is de fundamentele structurele en functionele eenheid van de eierstok en de ontwikkeling ervan is sterk gerelateerd aan de therapieën in de eierstok. Bloedvaten, voeding en hormonen aan de follikels leveren en dus spelen een belangrijke rol in de groei en de rijping van de follikels1.
Een combinatie van technologieën, met inbegrip van bloedvat selectieve markeringen, transgene muismodellen, en farmaceutische ontwikkeling, is onze kennis over ovariële vasculaire netwerken, angiogenese, en de functie van bloedvaten in gestegen folliculogenese. Het vruchtbeginsel is bekend als een actieve orgel omdat het hervormt verschillende weefsels en vasculaire netwerken tijdens folliculogenese en ovulatie. Dergelijke actieve remodelleren in de omvang en de structuur van schepen is vereist voor de biologische functie van de ontwikkeling en het werven van follikels.
Traditionele histologische en histomorphometric methodes gebruikend ovariële secties en immunolabeling van de bloedvaten zijn beperkt tot tweedimensionale (2D) afbeeldingen,2. Met de ontwikkeling van driedimensionale (3D) reconstructie technologieën, kunnen 2D beelden van weefsel segmenten overlappen zodat een 3D-structuur, maar deze methode heeft nog steeds enkele beperkingen — afdelen van het weefsel kan vernietigen de microstructuren, sommige delen van de weefsel ontbreken vaak, en belangrijke arbeid is betrokken bij het maken van 3D-reconstructies van de beelden verkregen uit segmenten. Geheel-weefsel 3D-beeldbewerking met confocale microscopie kan veel van deze beperkingen overwinnen, maar deze methoden zijn beperkt tot de evaluatie van de angiogenese in het embryonale eierstok3. Met behulp van hele weefsel clearing methoden zoals duidelijkheid4 kan verhogen het gevisualiseerde volume zodat deze problemen in de postnatale en volwassen eierstokken en dergelijke methoden bieden optische Goedkeuringvande het ovarium zonder eventuele structurele vervormingen. Beeldvorming van de 3D-architectuur van de eierstokken intact biedt een nauwkeurige afbeelding database voor analysesoftware van de afbeelding, zoals het Imaris softwarepakket gebruikt in dit werk.
Verbouwing van het ovarium tijdens volwassenheid is onderdeel van een dynamische fysiologische systeem, en dit maakt het ovarium een uitstekend model voor onderzoek naar de regulering van angiogenese. Evaluatie van de rol van ovariële bloedvaten in pathologische omstandigheden van het vrouwelijke reproductieve systeem zoals polycysteus ovarium syndroom of ovariële kanker kan bovendien worden bestudeerd door hele eierstokweefsel imaging. De ontwikkeling van de passieve duidelijkheid-methode en het gebruik van de software van de analyse van de geavanceerde beeld hebben gedetailleerde ruimtelijke informatie verstrekt over de relaties tussen de bloedvaten en ovariële structuren zoals follikels.

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	<tbody>
		<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>
</table>

three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary

Het vruchtbeginsel is het belangrijkste orgaan van het vrouwelijke voortplantingsstelsel en is essentieel voor de productie van de vrouwelijke gameten en voor de controle van het endocriene systeem, maar de complexe structurele relaties en driedimensionale (3D) therapieën platforms van de eierstok zijn niet goed beschreven. Om te visualiseren de 3D verbindingen en de architectuur van de bloedvaten in de intact eierstok, is de eerste belangrijke stap om de eierstok optisch duidelijk. Ter voorkoming van weefsel krimp, gebruiken we de hydrogel fixatie gebaseerde passieve duidelijkheid (duidelijk lipide-uitgewisseld Acrylamide-gekruist Rigid Imaging / immunokleuring/In situ-hybridisatie-compatibele weefsel Hydrogel) protocol van de methode om te wissen van een intact eierstok . Immunokleuring, geavanceerde multiphoton confocale microscopie en 3D-beeld-reconstructies werden vervolgens gebruikt voor de visualisatie van ovariële vaartuigen en folliculaire haarvaten. Met behulp van deze aanpak, we toonden een significante positieve correlatie (P < 0.01) tussen de lengte van de folliculaire haarvaten en het volume van de folliculaire muur.

Wij de passieve duidelijkheid methode aangepast in een snelle en eenvoudige methode voor passieve eierstok schakelen terwijl het behoud van de folliculaire en vasculaire architectuur en het verkrijgen van de hoogste fluorescent signaal van gelabelde markers van vaartuigen en follikels. De 3D-architectuur van de folliculaire therapieën werd bepaald door immunokleuring voor CD31, een marker voor endotheliale cellen6. CD31 vlekken in de eierstokken van volwassen muiz...

Watch this Scientific Journal Video about Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary at JoVE.com

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

Hier presenteren we een aanpassing van de passieve duidelijkheid en 3D reconstructie methode voor visualisatie van de ovariële therapieën en folliculaire haarvaten in de eierstokken intact muis.

Drie-dimensionale reconstructie van de vasculaire architectuur van de passieve duidelijkheid-gewist muis eierstok

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

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Microfabricage van Chip-en kleinbedrijf Stellingen voor drie-dimensionale celkweek

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

Hi-C: een methode om de driedimensionale architectuur van genoom-onderzoek.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

Generatie van Muizen Afgeleid van geïnduceerde pluripotente stamcellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Een analytisch instrument dat cellulaire morfologie Veranderingen van Drie-dimensionale Fluorescentie afbeeldingen kwantificeert

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

Met behulp van hoge resolutie Computed Tomography om de driedimensionale structuur en functie van Plant vaatstelsel Visualiseer

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Diavoorbereidingsmethode om driedimensionale chromatinearchitectuur van testiculaire kiemcellen te behouden

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolatie en Primaire Cultuur van de muis endotheelcellen

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

Isolatie van muizen coronaire vasculaire gladde spiercellen

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

FRET Imaging in Three-dimensional Hydrogels

FRET Imaging in Driedimensionale Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipo-Clear: Een weefsel Clearing methode voor driedimensionale beeldvorming van vetweefsel

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...