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; 2H2O, and 28.7 g of Na2HPO4 &#183;12H2O. Add dH2O to 1 L and stir ~2 h. Adjust to pH 7.4 with 1 N NaOH and store at room temperature. Dilute this 10x stock solution by 1/10 using dH2O and then make 0.1% Triton X-100 in PBS.</li>
		<li>Prepare the hydrogel solution (pH 7.5) by mixings 4% (wt/vol) paraformaldehyde (PFA) solution, 4% (wt/vol) acrylamide, 0.05% (wt/vol) bisacrylamide, and 0.25% (wt/vol) VA-044 initiator in double-distilled water on ice.</li>
		<li>Prepare the clearing solution (pH 8.5) by mixing 200 mM sodium borate buffer containing 4% (wt/vol) sodium dodecyl sulfate (SDS) in double-distilled water.</li>
	</ol>
	</li>
	<li>Transcardial perfusion and ovary preparation
	<ol>
		<li>Keep all solutions on ice during all procedures.</li>
		<li>Anesthetize adult female wild type C57BL/6 mouse with an intraperitoneal injection of pentobarbital (0.35 mg/kg) solution. Once it shows no toe-pinch response, the mouse is properly anesthetized.</li>
		<li>Use sticky tape to fix the mouse with limbs stretched out on a flat foam board.</li>
		<li>Expose the heart by an incision on the xiphoid-process through the chest, skin, and bones with scissors.</li>
		<li>Make an incision into the animal&#39;s right atrium, and then perfuse the left ventricle with 20 mL of ice-cold 1x PBS at 10 mL/min.</li>
		<li>Perfuse the animal with at least 20 mL of hydrogel solution at 10 mL/min.</li>
		<li>Incise the skin over the abdomen along the medioventral line with scissors, which exposes the whole enterocoelia, and remove the intestines. Collect the ovaries as well as the uterus to maintain the integrity of ovarian morphology.</li>
		<li>Remove the fat tissues around the ovary under a microscope.</li>
	</ol>
	</li>
	<li>Degassing
	<ol>
		<li>Place the ovaries in 10 mL hydrogel solution at 4 &#176;C for fixation for 3 days.</li>
		<li>Transfer the ovary into a 5-mL tube and fill the tube with hydrogel solution. Stretch parafilm over the top of the tube and then wrap parafilm around the neck of the tube. 
		Caution: Ensure that there are no bubbles between the parafilm and the hydrogel solution.</li>
		<li>Place the tube on a shaker in the incubator at 37 &#176;C for a maximum of 3 h to allow the hydrogel to polymerize.</li>
		<li>Remove the ovary from the gel using a spatula and carefully remove the excess gel from around the ovary by tissue paper. Wash the ovary four times with 1x PBS.</li>
	</ol>
	</li>
	<li>Passive clearing
	<ol>
		<li>Submerge the ovary in 10 mL of the clearing solution. Gently shake at 80 rpm at 37 &#176;C to start the clearing process.</li>
		<li>In the first week, change the solution every 3 days. After one week, refresh the clearing solution once a week until the ovary becomes clear. Usually this process takes about 2-3 weeks. 
		NOTE: The ovary can be directly immunostained or can be stored in sodium azide (0.02%) in PBS at 4 &#176;C for several weeks before immunostaining.</li>
	</ol>
	</li>
</ol>
2. Immunostaining
<ol>
	<li>Wash the ovary twice in PBST on the day 1 at 37 &#176;C on a shaker.</li>
	<li>Incubate the ovary on a shaker in primary antibodies/PBST solution (Table 1) on day 2-3 at 37 &#176;C. In the work presented here, the primary antibodies were platelet-endothelial cell adhesion molecule 1 (CD31) and tyrosine hydroxylase diluted at 1:10 and 1:50 in PBST, respectively. 
	Caution: If two or more kinds of primary antibody are used, make sure there are different animal sources.</li>
	<li>Wash off the primary antibodies with PBST buffer at 37 &#176;C on day 4 on a shaker.</li>
	<li>Incubate the ovary with the desired secondary antibodies in PBST on day 5-6 at 37 &#176;C on a shaker. In the work presented here, the secondary antibodies were Alexa Flour 488 for tyrosine hydroxylase and Alexa Flour 594 for CD31 diluted at 1:50 in PBST.</li>
	<li>Wash off the secondary antibodies with PBST buffer at 37 &#176;C on day 7 on a shaker.</li>
	<li>Transfer the tissue into refractive index matching solution for refractive index homogenization on day 8. Put the tissue on a paper with gridlines to check its visual clarity by checking whether the gridlines can be seen. Once the tissue has been fully clarified, it can be imaged.</li>
	<li>Make a putty cylinder that could just surrounds the tissue, and then shape it into a horseshoe shape and press the outer ridge tightly onto a glass slide in order to make a sealed space. The height of the putty should be a little taller than the ovary.</li>
	<li>Place the clarified ovary carefully into the center of the putty and add refractive index matching solution. Put a glass-bottom dish over the putty tightly and use putty to fix the dish.</li>
</ol>
3. Confocal Microscopy
<ol>
	<li>For imaging with a confocal microscope, in the &#34;Ni-E&#34; panel choose the lens (in this case water immersion 25X objective lens, 1.1-NA, 2 mm-WD) with the proper working distance (in this case 2.0 mm).</li>
	<li>In the &#34;Acquisition&#34; panel, select the number of channels. Here, use three channels.
	<ol>
		<li>First, use the &#34;eye port&#34; tab and the XY controller joystick to move the tissue to the center of field by checking through the microscope eyepieces.</li>
		<li>After turning off the shutter light, click the &#34;live&#34; tab to adjust the &#34;laser power&#34;, &#34;HV (Gain)&#34;, and &#34;offset&#39; for each channel separately. Higher laser power and HV can lead to higher signals, and higher offset can reduce the background noise.</li>
		<li>Select the proper &#34;Scan size&#34; and &#34;Count&#34;. Use a scan size of 1024 x 1024 &#181;m2 and a count of 4 to 8.</li>
	</ol>
	</li>
	<li>After setting the image quality for defining the depth of the tissue in the panel &#34;Z intensity correction&#34;, and after locating the lens on the top-most layer of the ovary (in the center of the tissue) and adjusting the acquisition for the top layer, click the &#34;plus&#34; button to define the top layer.
	<ol>
		<li>Move the lens down and set the appropriate HV and laser power for the bottom of the tissue and add the lowest layer information into the Z-intensity correction table. Click on &#34;To ND&#34; to synchronize the setting with the &#34;ND acquisition&#34; panel.</li>
	</ol>
	</li>
	<li>In the &#34;ND acquisition&#34; panel, first set the number of scanning steps. Then, tick the tabs &#34;Z&#34; and &#34;Large Image&#34;.
	<ol>
		<li>Go to the &#34;Scan large image&#34; window to define the size of the field according to the border of the tissue with the help of tissue visualization through the eyepieces. Go back to the &#34;ND acquisition&#34; panel to input the size of the scanning area (&#34;fields&#34;) and choose the overlap between 10% and 15%.</li>
		<li>Click &#34;Run Z correction&#34;, not &#34;Run now&#34;, to automatically set the proper HV, laser power, and offset for each layer, and wait for the image to process.</li>
	</ol>
	</li>
</ol>
4. 3D Reconstruction of Individual Follicles and Their Vasculatures
<ol>
	<li>First, use ImageJ5 to open the original NIS file. Click the &#34;Image&#34; button and choose the &#34;Color&#34; and the &#34;Split Channels&#34;. Then, click &#34;File&#34; and separately save the channels as tiff file series by using the &#34;Image Sequences&#34; option in &#34;Save as&#34;.</li>
	<li>Use Imaris to open one of the tiff file series. Click the &#34;Edit&#34; button and choose &#34;Add channels&#34; to add the rest of the channels. The name and color of the channel can be changed in the &#34;Display Adjustment&#34; tabs. The thickness of the tissue might need to be corrected in the &#34;Image Properties&#34; panel in the drop-down list under &#34;Edit&#34;.</li>
	<li>For the 3D cropping of the final images and removal of excess parts, click the &#34;Edit&#34; button and choose &#34;Crop 3D&#34;. The size of the field can be adjusted by dragging the border.</li>
	<li>To reconstruct the vessel signals with the Filaments algorithm in the Surpass panel, click the &#34;Filaments&#34; button to create a new Filament and click next.
	<ol>
		<li>In order to define the largest and the thinnest diameter, go to the &#34;Slice&#34; panel and find the tracing vessel. The distance will be automatically shown on the &#34;Measure&#34; panel by selecting two points at the maximum width of the vessel.</li>
		<li>Go back to the &#34;Surpass&#34; panel and input the measured width and click next.</li>
	</ol>
	</li>
	<li>Adjust the starting and seed point threshold. In the &#34;Camera&#34; panel, choose &#34;Select&#34;. If some of the automatically produced spheres need to be removed, press shift and click on the point. By choosing &#34;Navigate&#34; and moving the mouse, the structure can be rotated to ensure that all the correct points have been retained, and then click next.</li>
	<li>Choose the highest threshold for the local contrast and click next. Do not select &#34;Detect Spines&#34;, and continue to finalize the reconstruction of the blood vessel cylinder. The color can be edited by clicking the &#34;Color&#34; tab. The statistical data can be extracted in the &#34;Statistic&#34; panel of the reconstructed Filament. Excess parts can be removed in the &#34;Edit&#34; panel. 
	NOTE: Other algorithms can be used for measuring the volume of total follicles or follicular walls. In the work presented here, the oocytes were marked by the autofluorescence of both secondary antibodies. Because of the large amount of data, a workstation server was used for the data analysis.</li>
</ol>
5. Statistical Analysis
NOTE: Image analysis data are presented as means &#177; standard errors.
<ol>
	<li>Perform comparisons between the follicles using independent sample t-test, and use Pearson&#39;s correlation test to compare correlation coefficients of follicular wall and capillary length. Set a value of P &#60;0.05 as the limit for statistical significance.</li>
</ol>

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The authors have nothing to disclose.

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 ovarian architecture at different developmental stages.
To obtain 3D image data of intact tissues, the clearing process is the first important step. There are two main strategies &#8212; dehydration methods and hydrophilic reagent-based methods, such as passive CLARITY10. Because dehydration methods often lead to a decrease in fluorescent signals and often produce toxic organic solution waste, we have focused our attention on simplifying some steps of the passive CLARITY method for use with various tissue types.
The first critical procedure in the clearing process is the transcardial perfusion. During the perfusion, heart pumping plays an important role in the whole-body circulation of PBS and hydrogel solution, which can flush out the blood so as to decrease the background fluorescence. Thus, the perfusion surgery should be performed accurately and quickly to avoid the premature death of the animal before the hydrogel is completely distributed throughout the animal. All agents must be kept on ice during the entire experiment, as mentioned by Liang et al.11, or they will begin to polymerize before the perfusion is complete. The next important step is removing the hydrogel, and it is necessary to remove as much excess gel as possible because it will retain antibodies. Once the tissue is cleared, it should be removed from the clearing solution and stored in PBS at 4 &#176;C because too much time in the clearing solution will cause too much protein to be lost. In the confocal microscopy imaging, the Z-settings should always be set before the X-Y range. If the X-Y range is set down first, the Z-settings cannot be synchronized to the entire X-Y field.
Passive CLARITY was first introduced by Yang et al.12 In their protocol, they did not perform the perfusion step and instead degassed with nitrogen. In addition, their clearing solution did not include bisacrylamide in the hydrogel solution, they used 8% SDS in the clearing solution, and they used RIMS for refractive index matching media. This method was subsequently improved by increasing the SDS concentration13, adding the perfusion step and the nitrogen-free degassing procedure14, and combining the method with other clearing methods such as PARS-mPACT15. Here, we have further developed the protocol by combining transcardial perfusion and nitrogen-free degassing, decreasing the SDS in the clearing solution to 4%, and using FocusClear as the refractive index matching media, which provides better-quality microscopy images.
After passive CLARITY, confocal multiphoton microscopy and image analysis can be performed to obtain the intact 3D architecture of the ovarian follicular vasculature. This protocol transforms a non-light-permeable intact ovary into an optically transparent hydrogel-hybridized nanoporous form that is appropriate for penetration by macromolecules such as antibodies16. Unlike classical 2D sectional histological analyses, passive CLARITY and staining with specific markers allows the 3D mapping of relationships among capillaries and individual growing follicles.
In adult tissues, active vascular remodeling and angiogenesis mainly occurs during pathological processes such as wound healing, fracture repair, tumor and metastasis formations, retinopathies, fibroses, and rheumatoid arthritis17. However, cyclical changes during folliculogenesis represent a unique capacity for vasculature remodeling. After formation of the antrum in secondary follicles, follicular fluid fills it and surrounds the oocyte, which provides a suitable microenvironment for oocyte maturation and follicular development18. Previous 2D imaging studies showed that ovarian follicles are surrounded by a network of capillaries in the theca interna19. The granulosa layer is nonvascular, and each follicle is surrounded by its own capillaries. Consistent with the essential role of CD31 in vasculogenesis6, we found an increase in capillary length during folliculogenesis in the transition from primary to secondary follicles (Figures 2G and 2H). Instead of a random distribution of capillaries around the individual primary or secondary follicles, 3D reconstruction of capillaries demonstrated that capillary branches distributed in follicles were detectable in larger follicles, and in most cases these capillary branches formed a hollow space (Figure 3).
One major concern is that the adapted method takes a relatively long time to clear the ovary, but the long time is required for the best clearing effect. To save some time, we recommend collecting all of the needed samples at one time and clearing them together because the cleared tissues can be stored in PBS and 0.02% sodium azide at 4 &#176;C for several months. Another limitation is the working distance of the confocal microscope. Higher-magnification objective lenses have shorter working distances, which could be an obstacle for imaging of thick samples.
Several protocols have been introduced for the imaging of transparent and intact tissues, including BABB20, Scale21, 3DISCO22, ClearT23, SeeDB24, CLARITY16, passive CLARITY (with4 or without hydrogel25), PACT12, CUBIC26,27, FASTClear28, and FACT29, but only three of them have been used for whole ovarian tissue clearing and evaluation9,30,31. SeeDB30 and ScaleA231 have been introduced for imaging of whole ovarian tissues, but these protocols only allow a single immunostaining step. Passive CLARITY, however, allows for multiple protein-labeling reactions, as shown in the present study and in our previous study9.
In conclusion, our adapted protocol for determining the 3D structural relationship between follicular layers and blood vessels using whole clarified ovarian tissue provides a new approach for studying angiogenesis in different ovarian diseases such as polycystic ovary syndrome and ovarian cancers.

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 interrelationships between follicular capillaries and primary and secondary follicles (Figure 1).
The Spot transformation and Surface algorithms were used to detect the tyrosine hydroxylase-immunostained oocytes and granulosa and thecal layers and to calculate the total volume of follicles7,8. Because of the compaction of the follicles, some parts of cropped follicles were identified semi-manually. After Spot transformation of the individual follicular volume and oocytes and Surface reconstruction of the follicular wall layers and Filament reconstruction of the vessels, clear relationships between individual follicular vasculatures and the other measured indices, especially granulosa and thecal layer volumes, were observed (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56141/56141fig1.jpg" /> 
Figure 1: High-resolution imaging of an intact adult mouse ovary after clearing with the adapted passive CLARITY protocol. (A-B) Sections and XYZ large-scan acquisition. Three dimensional (3D) cropped secondary (C) and primary (D) follicles (top row) and 3D reconstructed capillaries, oocytes, and granulosa/thecal cells of the same follicle (bottom row) are shown as identified by the Filament, Surface, and Spot transformation algorithms. Scale bars = 200 &#181;m (A-B), 50 &#181;m (C), 30&#160;&#181;m (D).&#160; <a href="https://cloudflare.jove.com/files/ftp_upload/56141/56141fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56141/56141fig2.jpg" /> 
Figure 2: Comparative indices of the relationships between primary and secondary ovarian follicles and their vasculatures. The oocyte volume (A) was extracted after 3D reconstructions of follicular volume and oocytes in individual follicles as determined by the Spot transformation algorithm, and the follicular wall volume (B) was determined by the Surface algorithm and the capillaries were traced by the Filament algorithm. The follicular volume (C), the oocyte volume/capillary length ratio (D), the follicular wall volume/capillary length ratio (E), the follicular volume/capillary length ratio (F), the capillary length (H), and the correlation of capillary length and follicular wall volume (G) were calculated using the appropriate tools in the software package. Error bars represent the standard errors of the means. n = 6; * P &#60;0.05; ** P &#60;0.01. <a href="https://cloudflare.jove.com/files/ftp_upload/56141/56141fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56141/56141fig3.jpg" /> 
Figure 3: Three-dimensional reconstruction of the capillaries in secondary follicles of the mouse ovary. This reveals a hollow space above the oocyte side of the follicles (white squares show the borders of the space). <a href="https://cloudflare.jove.com/files/ftp_upload/56141/56141fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

three-dimensional-reconstruction-vascular-architecture-passive

Nanyang Technological University

Research

JoVE Journal

Biology

8.6K Views.  Fudan University. 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.

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

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

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

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

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

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

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

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

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

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

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

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

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

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

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

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 employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

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

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

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

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple&#160;checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.
In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.
This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

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

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Chapters in this video

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Isolation of Murine Coronary Vascular Smooth Muscle Cells

FRET Imaging in Three-dimensional Hydrogels

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

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing