Name: whole mount immunofluorescence and follicle quantification of cultured mouse ovaries
Uploaded: 2018-05-02T14:45:00
Description: Watch this Scientific Journal Video about Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries at JoVE.com

We thank Rebecca Williams and Johanna Dela Cruz from the Cornell BRC Imaging and Andrew Recknagel for helpful suggestions and technical assistance. This work was supported to National Institutes of Health grant S10-OD018516 (to Cornell&#39;s Imaging Facility), T32HD057854 to J.C.B. and R01-GM45415 to J.C.S.

Cornell University&#39;s Institutional Animal Care and Use Committee (IACUC) has approved all the methods described here, under protocol 2004-0038 to JCS.
1. Preparation of Instruments and Culture Media
<ol>
	<li>Wipe the working area clean with 10% bleach and allow the bleach to remain on the work area surface for at least 5 min. After 5 min, remove the excess bleach with clean paper towels and 70% ethanol (EtOH). Clean the dissecting microscope thoroughly with 70% EtOH. 
	NOTE: It is ...

<ol>
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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> Biology of Reproduction. 93 (5), (2015).</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> Scientific Reports. 7, (2017).</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=ScaleS:+an+optical+clearing+palette+for+biological+imaging.">ScaleS: an optical clearing palette for biological imaging.</a> Nature Neuroscience. 18 (10), 1518-1529 (2015).</li><li>Ke, M. -. 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The study of mammalian reproduction requires using and quantifying specialized cells that are not routinely amenable to cell culture. However, ex vivo culture systems are effective at maintaining ovary and follicle viability1,15. During ovary culture, the tissue requires a larger surface area for exchange of nutrients through diffusion. Therefore, 5-day old mouse ovaries are ideal in size and shape for organ culture. This protocol was optimized for ovari...

In order to study the cellular composition and morphological features of mammalian ovaries, scientists often rely on in vivo experiments followed by immunohistological staining of paraffin embedded ovaries. More recently though, whole ovary organ culture has proven to be an effective alternative to study ovarian function1,2,3,4 because the technique can be coupled with better visualization and quantification tools. Traditionally, analysis of ovarian morphology depends on reconstructing three-dimensional ovarian architecture from paraffin embedded serial sections, but, in addition to being laborious and time consuming, serially sectioning paraffin embedded tissue does not guarantee proper reconstruction of the organ, and sections are often lost or mis-ordered in the process.
In addition to the technical challenges associated with serial sectioning, there are also variations in the methods routinely used to quantify follicle numbers per ovary5,6. The methodological variability currently used impairs meta-analysis of ovarian reserve across studies5,7. For example, follicle numbers from different research articles can vary by 10-fold or more between similar developmental ages within a specific strain6. These large differences in reported follicle quantification can lead to confusion and have hindered cross-study comparisons. Experimentally, traditional approaches to follicle quantification from serial sections are performed by counting follicles of a pre-defined number of sections (e.g., every fifth, tenth, or other section). Variability in follicle counts using this approach arises not only from the periodicity in which sections are counted but also from variations in section thickness, and technical experience in generating serial sections5,6. In addition to its variability, another disadvantage of traditional tissue sectioning is that the sectioning of small ovaries from young animals is especially challenging and highly dependent on tissue orientation8.
The protocol below describes a routinely used ovary culture technique1 but greatly improves upon traditional follicle quantification by substituting physical sectioning with tissue clearing and optical sectioning using confocal microscopy8,9. Clearing using tissue immersion (without the need for transcranial perfusion or electrophoresis) in a urea- and sorbitol-based solution (e.g., ScaleS(0)10) proved compatible with the immunostaining and allowed for the reduction of clearing time without compromising depth of imaging. Other reported methods (e.g., ScaleA28,10, SeeDB11, ClearT12, and ClearT212) are either more time consuming or do not allow in-depth optical resolution of the sample. Optical sectioning is advantageous because it is less labor intensive and maintains the organ&#39;s three-dimensional architecture7,8. Another benefit of this approach is that preparation of the samples does not require costly reagents to clear the tissue and can be conducted with relative ease.
Specifically, the protocol described has been optimized for cultured mouse ovaries at postnatal day five but has been conducted on ovaries ranging from postnatal day 0 - 10. The method makes use of an ovary culture system in which the tissue naturally attaches to the membrane on which it is cultured, facilitating organ handling and manipulation. The culture system described can be used to maintain explanted ovaries for up to 10 days and to assess how different experimental conditions may interfere with oocyte survival13. The quantification procedure described is performed using the non-commercial image processing package FIJI-ImageJ14 and can be conducted on most personal computers. Furthermore, images used for quantification can be made widely available for the scientific community, thus allowing for future meta-analysis.

<table border="0" cellpadding="0" cellspacing="0" width="882">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Micro dissecting scissor 4.5&#34;, straight, sharp points</td>
			<td style="width:200px;">Roboz</td>
			<td style="width:117px;">RS-5912</td>
			<td style="width:224px;">Micro dissecting scissor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Jewelers style forceps 4-3/8&#34;, style 5</td>
			<td>Integra Miltex</td>
			<td>17-305X</td>
			<td>Fine tip forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Iris Scissors 4&#34;, straight, sharp points</td>
			<td>Integra Miltex</td>
			<td>18-1618</td>
			<td>Micro dissecting iris scissor or micro dissecting spring scissor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Minimal Essential Media (MEM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11090-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serium</td>
			<td>Corning</td>
			<td>35011CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td>Used in Step 1.3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35mm Tissue Culture Treated Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Nunc&#8482; 24-Well Carrier Plate for Cell Culture Inserts</td>
			<td>ThermoFisher Scientific</td>
			<td>141006</td>
			<td>Pore size: 8&#956;m</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>16% solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">1X Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nutator mixer GyroTwister&#8482;</td>
			<td>Labnet</td>
			<td>S1000-A-B</td>
			<td>three dimensional shaker</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Normal Goat Serum</td>
			<td>VWR</td>
			<td>103219-578</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Bovine Serum Albumen (BSA)</td>
			<td>VWR</td>
			<td>97061-416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sodium Azide (NaN3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium borohydride solution (NaBH4)</td>
			<td>Sigma-Aldrich</td>
			<td>452904-25ML</td>
			<td>Use the solution, rather than the tablet or powder form&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvinyl Alcohol (PVA)</td>
			<td>Sigma-Aldrich</td>
			<td>P8136-250G</td>
			<td>Cold water soluble</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton&#8482; X-100</td>
			<td>Sigma-Aldrich</td>
			<td>93443-100ML</td>
			<td>polyethylene glycol tert-octylphenyl ether</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters</td>
			<td>ThermoFisher Scientific</td>
			<td>725-2520</td>
			<td>25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10mL Syringes</td>
			<td>BD</td>
			<td>309695</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-p63 antibody (4A4)</td>
			<td>Biocare Medical</td>
			<td>CM 163A</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-MVH antibody</td>
			<td>Abcam</td>
			<td>ab13840</td>
			<td>Dilution 1:600</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor&#174; goat anti-mouse 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11032</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor&#174; goat anti-rabbit 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11034</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4,6-Diamidino-2-phenylindole (DAPI)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(-)sorbitol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>240850-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G9012-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5378-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650-5X10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FIJI-ImageJ</td>
			<td></td>
			<td></td>
			<td>Image processing software</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Disposable plastic transfer pipettes</td>
			<td>VWR</td>
			<td>414044-034</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole mount immunofluorescence and follicle quantification of cultured mouse ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

This protocol includes 6 major steps following dissection of the ovaries, as outlined in Figure 1. Figure 2, Figure 3, Figure 4 highlight the most novel features of this protocol, which include optimization of tissue clearing for ovaries and whole tissue oocyte quantification using FIJI-ImageJ. Figur...

Watch this Scientific Journal Video about Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries at JoVE.com

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

The overall goal of this procedure is to obtain images of cultured whole ovaries that are compatible with automated follicle quantification. This method can help answer key questions in the reproductive biology field such as how different conditions interfere with ovarian fitness. The main advantage of this technique is that it is less labor intensive and maintains the organ's three-dimensional architecture.Though this method has been optimized for cultured mouse ovaries at postnatal day five, it can also be applied to other ages and tissues such as embryonic gonads or young mice testes. Start with a clean work surface. Wipe with ten percent bleach and allow the bleach to remain on the work area surface for at least five minutes.After five minutes, remove excess bleach with clean paper towels and 70 percent ethanol. Clean the dissecting microscope thoroughly with 70 percent ethanol and place a dry paper towel on the stage. Wrap clean forceps and scissors with a paper towel dampened with 70 percent ethanol.Then make 50 milliliters of ovary culture media in a conical tube and warm in a 37 degrees Celsius water bath. After the medium has warmed, prepare plates and inserts by first adding one milliliter of ovary culture medium to 35 millimeter tissue culture plates. Then add approximately 0.55 milliliters of medium to the wells of a 24-well tissue culture plate and place an insert into each well containing medium.Make sure that the membranes touch the media. Place the 35 millimeter plates and the 24-well carrier plate into a 37 degrees Celsius, five percent carbon dioxide and atmospheric oxygen incubator. Then place a hot plate next to the dissection scope and set the temperature to 37 degrees Celsius.If the tissue culture incubator is not close to the dissection area, place one of the 35 millimeter tissue culture dishes containing ovary culture media on the hot plate. Then begin the dissection by spraying the freshly euthanized female with 70 percent ethanol and place on top of the dry paper towel under the dissection microscope. After making an incision in the abdominal cavity, extrude the intestines and locate the ovaries.Extract the ovaries and place them into the culture medium in the 35 millimeter dish. Once the ovaries have been dissected, increase the magnification of the dissection microscope to better visualize ovaries and any attached tissue. With clean fine tip forceps, remove all non-ovarian tissue such as bursa and fatty tissue without damaging the ovaries.Next, place each pair of ovaries onto a pre-soaked cell culture insert and adjust the volume of the medium to ensure that it is sufficient to keep the organ moist without completely submerging it. Place explanted organs in a 37 degrees Celsius, five percent carbon dioxide and atmospheric oxygen incubator. At the desired experimental time point, remove the ovary culture media from the plate and fix the cultured ovaries on the insert by covering the tissue with freshly prepared four percent PFA solution.Seal the edges of the plate with paraffin film to avoid evaporation and place the samples at four degrees Celsius overnight. Rinse the tissue three times with 70 percent ethanol. Then store in scintillation vials filled with 70 percent ethanol at four degrees Celsius until further processing.Begin staining by removing the 70 percent ethanol from the vials and washing the fixed tissue with PBS at room temperature with shaking for a minimum of four hours. After removing the PBS, add enough permeabilization solution to completely submerge the ovaries. Place on an orbital shaker and incubate at room temperature for four hours.Replace the permeabilization solution with enough blocking solution to completely submerge the ovaries. Then incubate the sealed vials at room temperature for a minimum of 12 hours on an orbital shaker. After blocking, place the inserts into a 24-well plate and add about 750 microliters of primary antibody solution ensuring that ovaries are completely submerged.Then place the sealed plate on the orbital shaker and incubate for four days at room temperature. After four days, remove the primary antibody and add a generous amount of washing solution. Wash the ovaries overnight.The next day, replace with fresh washing solution and incubate on the orbital shaker for two hours or more. After the washes, add secondary antibody solution. Protect the samples from light and incubate on an orbital shaker at room temperature for two days.After two days, remove the secondary antibody solution from the samples and add washing solution. Begin by preparing ScaleS0 clearing solution. Mix the solution by inversion and incubate at 50 degrees Celsius for 30 minutes.De-gas the ScaleS0 clearing solution. Then remove the washing solution from the immunostained ovaries. Add the clearing solution.Cover the plate with aluminum foil and return it to the orbital shaker. It is extremely important to be patient during the staining and clearing steps. Once the tissue has become transparent, use a fine tip scalpel to carefully remove the insert membranes containing the cultured ovaries.Place the sample on a glass slide and gently cover the sample with a coverslip. Then proceed to image the samples on a microscope capable of optical sectioning. This image shows a postnatal day five ovary which has not been cleared.Here, the ovary is shown one hour after the addition of clearing solution. The ovary will begin to become translucent within minutes of being submerged in clearing solution. Optical sectioning of cultured ovaries without clearing is possible, but cells deep within the tissue have a signal that is difficult to differentiate from background, which impedes proper oocyte quantification.In contrast, this representative image shows a cleared sample in which oocyte quantification throughout the entire organ is possible. While attempting this procedure, it's important to remember that antibodies have to diffuse through the tissue;thus, thicker tissues will require longer incubation time. After watching this video, you should have a good understanding of how to culture, stain, and image whole ovaries.The text describes the use of a free software, Fiji ImageJ, that can be used for simple follicle quantification. Don't forget that working with paraformaldehyde and sodium azide can be hazardous and precautions such as use of fume hoods and personal protective equipment should always be taken while performing this procedure.

Research

JoVE Journal

Developmental Biology

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

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

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

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

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

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