Name: organ culture and whole mount immunofluorescence staining of mouse wolffian ducts
Uploaded: 2017-01-13T15:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts at JoVE.com

We would like to thank members of the gynaecology oncology group for critical reading of this manuscript. This work is in part supported by funding from the National Health and Medical Research Council, the Australian Research Council, and the Cancer Institute NSW (P.S.T). M.K. is a recipient of the University of Newcastle Postgraduate Research Fellowship.

Animal care and experimental procedures were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of the University of Newcastle and confirmed to the New South Wales Animal Research Act, New South Wales Animal Research Regulation, and the Australian code for the care and use of animals for scientific purposes. All the procedures undertaken on mice were approved by the Animal Care and Ethics Committee of the University of Newcastle.
1. Time Mating
<ol>
	<li>Pair 6 - 8 weeks old male and female mice just prior to the end of daylight cycle.</li>
	<li>Check the females for the presence of vaginal plugs early morning (on or before 8.00 AM), every day. Day of plug is considered as 0.5 dpc.</li>
	<li>Transfer the female with vaginal plug into a new cage (without male) and label the date of plug.</li>
</ol>
2. Isolation of Mouse Embryonic Gonadal Ridges
<ol>
	<li>Isolate mouse embryonic gonadal ridges from 15.5 dpc&#160;pregnant females as described in 9, with a few modifications as described below.</li>
	<li>Carry out dissection before noon as the time pregnant females are considered to enter into the next day of pregnancy&#160;by afternoon.</li>
	<li>Sacrifice 15.5 dpc&#160;pregnant females using cervical dislocation (or as per the approved protocol by the institutional Animal Ethics Committee).</li>
	<li>Keep the mouse on its back on absorbent tissue paper and spray the ventral side of the abdomen with 70% ethanol.</li>
	<li>Lift the lower abdominal skin (ventral side) with forceps and make a lateral incision using surgical scissors, extending from urogenital opening to the end to rib cage. Pull the skin towards the head of the animal to expose the abdomen.</li>
	<li>Cut the abdominal muscles with surgical scissors to expose peritoneal cavity. Grab the gravid uterus with forceps just above the cervix and make a cut. Now lift the uterus by holding on to the urinary bladder and cut the uterine broad ligament followed by cuts at the uterotubal junctions to detach uterus from the peritoneal attachment.</li>
	<li>Transfer the uterus into a 50 mL tube containing ice-cold HBSS (Hank&#39;s Balanced Salt Solution). Wash the gravid uterus gently with ice-cold HBSS to remove the excess blood.</li>
	<li>Keep the uterus in a Petri dish containing ice-cold HBSS and keep on ice. Cut the uterine wall and take out the embryos. Keep the embryos in a fresh Petri dish with HBSS on ice.</li>
	<li>Take a clean sterile tissue paper and put it on a new Petri dish. Spray 70% ethanol on the tissue paper. Keep embryo on this tissue paper on the lateral side and cut it from lower abdomen using a sterile blade in the direction as shown by dotted line in Figure 1.</li>
	<li>Fix the embryo on a sterile sponge base by pinning the vertebral column.</li>
	<li>Place it under the dissecting stereomicroscope and carefully cut along the ventral midline. Remove the liver and intestines. The mouse urogenital system (kidney, ureter, testis, WD, and vas deferens) should now be visible.</li>
	<li>Cut out the testis and WD, and keep in a fresh Petri dish with HBSS, on ice. To take out the WD and testis, cut the vas deferens close to its attachment to urethra and cut the attachment of lower portion of WD from gubernaculum.</li>
	<li>Pool the testes and WDs from the embryos of the same group (this may vary as per the experimental plan).</li>
</ol>
3. Culture of Embryonic Gonadal Ridges (Figure 2)
<ol>
	<li>Prepare medium for culturing the embryonic gonadal ridges by supplementing DMEM/F12 with 10% FBS (Fetal Bovine Serum), 1% penicillin/streptomycin and 1% L-glutamine. Warm the media to 37 &#176;C in a water bath.</li>
	<li>Add 300 &#181;L of media per well of a 24-well cell culture plate.</li>
	<li>Take a fresh Petri plate and put a small drop of sterile HBSS on it. Put a polycarbonate track etch membrane (0.8 &#181;m) on this drop of HBSS with its&#160;shining surface facing the HBSS.</li>
	<li>Using clean forceps put two WDs and gonads on the membrane (on its rough surface, facing upward) and remove the maximum amount of HBSS using sterile wipes/absorbent paper. Do not touch the tissue with absorbent paper; otherwise, it will damage the tissue.</li>
	<li>Ensure that neither the WDs nor the gonads are touching each other. Otherwise, they will stick together during subsequent incubation.</li>
	<li>Transfer the membrane with the tissues in the well of a 24-well plate containing 300 &#181;L culture medium and culture them at air-medium interface. Too much culture medium on membranes results in a cystic growth of WDs.</li>
	<li>Incubate the plate at 37 &#176;C incubator, in the presence of 5% CO2.</li>
	<li>Change the culture medium every day. To change the medium with the pipette, remove the medium from the well first and then add 300 &#181;L of the prewarmed fresh medium. 
	Note: For studying the effect of different signaling pathways on WD morphogenesis, chemical activators and/or inhibitors at the required concentrations can be added to the culture medium.</li>
	<li>Culture the tissues for 3 d. Within 3 d, uncoiled WDs collected from a 15.5 dpc&#160;embryo transform into highly convoluted tubes (Figure 3B). The addition of the Wnt inhibitor, IWR1,&#160;results in inhibition of WD coiling (Figure 3C).</li>
	<li>For harvesting these tissues take a Petri dish with ice-cold PBS. Transfer the membrane with cultured gonad and WD into the Petri plate. The membrane will float on the PBS. Press the membrane to sink it in PBS and take out the gonad and WD.</li>
	<li>Fix the tissues with 4% paraformaldehyde (PFA) O/N at 4 &#176;C or for 1 h at RT. If needed, take bright field images of the WDs before fixation.</li>
</ol>
4. Whole Mount Immunofluorescence
<ol>
	<li>Wash the fixed tissues 3x with PBS-T (PBS + 1% Triton X-100) with slow rocking, 10 min each, at RT.</li>
	<li>Dehydrate the tissues in a graded series of ethanol (25%, 50%, 75% and 100%), 10 min each, at 4 &#176;C, with slow rocking (~30 rpm). At this step, the tissues can be stored at 4 &#176;C in 75% ethanol.
	<ol>
		<li>To change the ethanol, keep the tissues undisturbed for 2 min and let the tissues settle down. Take out as much supernatant as possible and then add the next higher concentration of ethanol. To avoid any damage to the tissues, the pipette tip should not touch the tissues at any stage.</li>
	</ol>
	</li>
	<li>Following dehydration, rehydrate the tissues in a graded series of ethanol (100%, 75%, 50% and 25%), 10 min each, at 4 &#176;C, with slow rocking.</li>
	<li>Wash the tissues with PBS-T (PBS + 0.1% Triton X-100), 4x, 20 min each, at RT, with gentle rocking (~30 rpm).</li>
	<li>Block the tissues for 1 h at RT with blocking buffer (PBS + 1% BSA + 0.2% non-fat dry milk powder + 0.3% Triton X-100).</li>
	<li>After blocking, transfer the tissues to primary antibody solution (diluted in blocking buffer) and incubate O/N&#160;at 4 &#176;C with gentle rocking (~30 rpm). Dilutions of the primary antibodies used are described in the Materials Table.</li>
	<li>The next day, wash tissues with PBS-MT (PBS + 2% non-fat dry milk powder + 0.5% Tween-20), 4x, 30 min each, at RT, with gentle rocking (~30 rpm).</li>
	<li>Following this washing step, add secondary antibody at a dilution of 1:250 (this step needs optimization for individual antibodies) in blocking buffer and incubate for 1 h at RT, with gentle rocking.</li>
	<li>From now onwards keep the tissues covered with aluminum foil to prevent exposure to light as secondary antibodies are labeled with light sensitive fluorophores.</li>
	<li>Wash the tissues 3x with PBS-T (PBS + 0.1% Tween-20), 30 min each, with gentle&#160;rocking.</li>
	<li>Prepare slides for mounting the stained tissues.
	<ol>
		<li>For this, cut the glass cover slips into thin strips using a diamond pen and stick two strips one over the other on the glass slide using nail enamel. Make two boundaries and place tissues in between these two boundaries.</li>
		<li>Add DAPI containing mounting medium and put on a cover slip. Apply nail enamel at the corners of the cover slip to fix it to the slide. 
		Note: Tissues are now ready for imaging. Store these slides at -20 &#176;C.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Joseph, A., Yao, H., Hinton, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+morphogenesis+of+the+Wolffian/epididymal+duct,+more+twists+and+turns.">Development and morphogenesis of the Wolffian/epididymal duct, more twists and turns.</a> Dev Biol. 325, 6-14 (2009).</li><li>Murashima, A., Xu, B., Hinton, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+normal+and+abnormal+development+of+the+Wolffian/epididymal+duct+by+using+transgenic+mice.">Understanding normal and abnormal development of the Wolffian/epididymal duct by using transgenic mice.</a> Asian J Androl. 17, 1-7 (2015).</li><li>Carroll, T. J., Park, J. S., Hayashi, S., Majumdar, A., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt9b+plays+a+central+role+in+the+regulation+of+mesenchymal+to+epithelial+transitions+underlying+organogenesis+of+the+mammalian+urogenital+system.">Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system.</a> Dev Cell. 9, 283-292 (2005).</li><li>Kumar, M., Syed, S. M., Taketo, M. M., Tanwar, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+Wnt/&#946;catenin+signalling+is+essential+for+epididymal+coiling.">Epithelial Wnt/&#946;catenin signalling is essential for epididymal coiling.</a> Dev Biol. 412, 234-249 (2016).</li><li>Murashima, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+roles+of+androgen+signaling+in+Wolffian+duct+stabilization+and+epididymal+cell+differentiation.">Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation.</a> Endocrinology. 152, 1640-1651 (2011).</li><li>Lin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+limitations+of+genetically+modified+mouse+models+in+drug+discovery+and+development.">Applications and limitations of genetically modified mouse models in drug discovery and development.</a> Curr Drug Metab. 9, 419-438 (2008).</li><li>Rak-Raszewska, A., Hauser, P. V., Vainio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+In+Vitro+Culture:+What+Have+We+Learned+about+Early+Kidney+Development?.">Organ In Vitro Culture: What Have We Learned about Early Kidney Development?.</a> Stem Cells Int. 959807, 1-16 (2015).</li><li>Hirashima, T., Adachi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedures+for+the+Quantification+of+Whole-Tissue+Immunofluorescence+Images+Obtained+at+Single-Cell+Resolution+during+Murine+Tubular+Organ+Development.">Procedures for the Quantification of Whole-Tissue Immunofluorescence Images Obtained at Single-Cell Resolution during Murine Tubular Organ Development.</a> PLoS One. 10, 0135343 (2015).</li><li>Chan, W. Y., Blomberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+Development:+Methods+and+Protocols.">Germline Development: Methods and Protocols.</a> Methods Mol Biol. 825, (2012).</li><li>Karner, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tankyrase+Is+Necessary+for+Canonical+Wnt+Signaling+During+Kidney+Development.">Tankyrase Is Necessary for Canonical Wnt Signaling During Kidney Development.</a> Dev Dyn. 239, 2014-2023 (2010).</li><li>Traykova-Brauch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+versatile+system+for+acute+and+chronic+modulation+of+renal+tubular+function+in+transgenic+mice.">An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.</a> Nat Med. 14, 979-984 (2008).</li></ol>

The authors have nothing to disclose.

The organ culture system has many advantages over the traditional cell culture system. This system retains the original structural relationships and interactions between different cell types. It mimics in vivo systems and gives more accurate information than cell culture studies where the niche factor is absent. The use of organ culture systems even have advantages over using the whole animal. These include the higher cost of using the whole animal, easy care and maintenance of organ culture system, etc.</em...

The male reproductive system primarily consists of testis, the site for germ cell development and differentiation, and a complex ductal system that is required for the maturation, transport, and storage of sperm. Epididymis is a tubular organ that connects testis with vas deferens and mainly involved in post-testicular development and maturation of germ cells 1. The highly coiled adult mouse epididymis develops from a simple and straight precursor tube, Wolffian duct (WD) 1. The complex and coiled structure of epididymis is essential for sperm to acquire the capacity to fertilize female germ cells 2. How such an essential organ for male fertility develops and gets into shape is not well understood. Various signaling pathways have been shown to be involved in the development of WD such as the Wnt signaling pathway 3,4 and the androgen signaling pathway 5. Our recent work has established the requirement of balanced Wnt signaling&#160;for WD coiling during prenatal development 4. However, further studies are required to understand the mechanisms involved in postnatal epididymal coiling, cellular differentiation, and cell-to-cell communication within the epididymal epithelium and with interstitial cells.
Genetically modified mouse models have been proven to be a powerful tool for identification/validation of molecular mechanisms underlying development and disease of various organ systems 6. Despite its extensive use, there are significant limitations of genetically modified mouse models, including the unpredicted phenotype of targeted mouse mutants with regards to presumed gene function, and no phenotype in null mutants in some models, influence of genetic and environmental factors, labor intensive and time-consuming process of developing mouse models. Therefore, the interpretation of the significance of the findings only from genetically modified mouse models is not always straightforward 6. These limitations can be overcome by using in vitro organ culture system which gives us the flexibility to manipulate multiple signaling pathways in real time in controlled culture conditions. Organ culture system is instrumental in understanding the physiology and pathology of whole organs 7. Moreover, imaging of whole organs stained with fluorophore labeled antibodies enables us to visualize the markers of interest in a three-dimensional context, as it exists in vivo, thereby providing a better understanding of the organ&#39;s shape, structure, and function 8.
Here, we have described the methods for isolation of mouse embryonic gonadal ridges, in vitro culture&#160;and whole mount immunofluorescence imaging of WDs, that can be applied to answer a variety of questions concerning the morphogenesis of tubular organs such as WDs. In this protocol, we isolated mouse embryonic urogenital ridges from 15.5 days post coitum (dpc) pregnant dams and cultured them for 3 d&#160;followed by immunostaining for an epithelial cell marker (cytokeratin 8, CK8), a cell proliferation marker (phospho-Histone 3, PH3) and active &#946;catenin.

<table><tbody><tr><td>24-well plates</td><td>Corning, USA</td><td>3524</td><td>can be purchased from other vendors</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>GE Healthcare Life Sciences, USA</td><td>SH30031.02</td><td>can be purchased from other vendors</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)</td><td>Sigma, USA</td><td>D8437</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Interpath,&amp;nbsp; Australia</td><td>SH30034.02</td><td>10% FBS used in culture medium</td></tr><tr><td>L-Glutamine</td><td>GE Healthcare Life Sciences, USA</td><td>SH30034.01</td><td>1% concentration used</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco, USA</td><td>15070-063</td><td>1% concentration used</td></tr><tr><td>Whatman Nuclepore Polycarbonate Track-Etch&amp;nbsp;</td><td>Whatman, USA</td><td>110409</td><td>Membrane (0.8 &amp;micro;m)</td></tr><tr><td>IWR1</td><td>Sigma, USA</td><td>10161-5mg</td><td>100 &amp;micro;M concentration used</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences, USA</td><td>15710</td><td>16% stock, 4% in PBS for use. TOXIC - wear gloves and cannot&lt;br/&gt; be disposed of in the sink</td></tr><tr><td>Ethanol</td><td>Thermo Scientific Fisher, USA</td><td>214-20L PL</td><td>Absolute, make 25, 50 and 75% with milliQ water.</td></tr><tr><td>Tween-20</td><td>Thermo Scientific Fisher, USA</td><td>2509-500ml</td><td>Very viscous, careful while dispensing</td></tr><tr><td>Triton X-100</td><td>Sigma, USA</td><td>T8787-50ml</td><td>Very viscous, careful while dispensing</td></tr><tr><td>Di-Sodium Hydrogen Phosphate</td><td>Thermo Scientific Fisher, USA</td><td>621-500mg</td><td>can be purchased from other vendors</td></tr><tr><td>Sodium Di-hydrogen Phosphate</td><td>Ajax Finechem, USA</td><td>4745-500g</td><td>can be purchased from other vendors</td></tr><tr><td>Sodium Chloride</td><td>Thermo Scientific Fisher, USA</td><td>465-500g</td><td>can be purchased from other vendors</td></tr><tr><td>Cytokeratin8/Troma I</td><td>Developmental Studies Hybridoma Bank, USA (DHSB)</td><td>TROMA-I</td><td>1:250 in blocking buffer</td></tr><tr><td>active &amp;beta;catenin&amp;nbsp;</td><td>Cell Signalling Technology,&amp;nbsp; USA</td><td>D13A1</td><td>1:200 in blocking buffer</td></tr><tr><td>phospho-Histone 3</td><td>Millipore, MA, USA</td><td>06-570</td><td>1:200 in blocking buffer</td></tr><tr><td>Alexa488 Goat anti-rabbit IgG</td><td>Jackson ImmunoResearchLabs,&amp;nbsp; USA</td><td>111-545-047</td><td>1:250 in blocking buffer</td></tr><tr><td>Alexa594 Goat anti-Rat IgG</td><td>Jackson ImmunoResearchLabs,&amp;nbsp; USA</td><td>112-585-072</td><td>1:250 in blocking buffer</td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific, USA</td><td>242-500ml</td><td>can be purchased from other vendors</td></tr><tr><td>n-Propyl galate</td><td>Sigma, USA</td><td>2370</td><td>-</td></tr><tr><td>2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI)</td><td>Sigma, USA</td><td>D9564-10mg</td><td>Use to stain nucleic acids (DNA)</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Thermo Fisher Scientific, USA</td><td>2225-500ml</td><td>Solvent</td></tr><tr><td>Cover Slips (24 x 50 mm)</td><td>Lomb Scientific</td><td>CS24501GP</td><td>-</td></tr></tbody></table>

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.

During development, the WD undergoes significant changes wherein a simple and straight tube is transformed into a highly complex and coiled duct. Using methods described above, WDs undergo a similar transformation in culture conditions. Here we have shown the results from WDs cultured for 3 d&#160;(Figure 3). To dissect molecular mechanisms involved in WD morphogenesis, the culture medium can be supplemented with inhibitors and activators targeting different signaling pathways. Figure 3C...

Watch this Scientific Journal Video about Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts at JoVE.com

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

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 ...

organ-culture-whole-mount-immunofluorescence-staining-mouse-wolffian

Research

JoVE Journal

Developmental Biology

10.7K Views.  University of Newcastle. 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.

Video: Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Cancer Research

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

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.

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, ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Neuroscience

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Biology

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Summary

Explore More Videos

Chapters in this video

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology