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.

whole-mount-immunofluorescence-follicle-quantification-cultured-mouse

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 within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between 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 distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

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

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

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

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 integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

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

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 optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

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

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

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

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

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

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

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 and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

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 the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

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 heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

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

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

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