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

Vi tackar Rebecca Williams och Johanna Dela Cruz från den Cornell BRC Imaging och Andrew Recknagel för användbara förslag och tekniskt bistånd. Detta arbete fick stöd till National Institutes of Health grant S10-OD018516 (till Cornells Imaging anläggning), T32HD057854 till J.C.B. och R01-GM45415 till J.C.S.

Cornell Universitys institutionella djur vård och användning kommittén (IACUC) har godkänt alla de metoder som beskrivs här, enligt protokoll 2004-0038 till JCS.
1. beredning av instrument och kultur Media
<ol><li>Torka av arbetsområdet rent med 10% blekmedel och tillåta lut kvar på ytan med arbete området i minst 5 min. Efter 5 min, ta bort överflödig lut med rent hushållspapper och 70% etanol (EtOH). Rengöra mikroskopet dissekera noggrant med 70% EtOH. Obs: Det är viktigt ...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+follicle+counts+-+not+as+simple+as+1,+2,+3.">Ovarian follicle counts - not as simple as 1, 2, 3.</a> Reproductive Biology and Endocrinology. 1, 11 (2003).</li><li>Bucci, T. J., Bolon, B., Warbritton, A. R., Chen, J. J., Heindel, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+sampling+on+the+reproducibility+of+ovarian+follicle+counts+in+mouse+toxicity+studies.">Influence of sampling on the reproducibility of ovarian follicle counts in mouse toxicity studies.</a> Reproductive Toxicology. 11 (5), 689-696 (1997).</li><li>Skodras, A., Marcelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-Generated+Ovaries+to+Assist+Follicle+Counting+Experiments.">Computer-Generated Ovaries to Assist Follicle Counting Experiments.</a> PLOS ONE. 10 (3), e0120242 (2015).</li><li>Malki, S., Tharp, M. 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. -. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nature Neuroscience. 16 (8), 1154-1161 (2013).</li><li>Kuwajima, T., Sitko, A. A., Bhansali, P., Jurgens, C., Guido, W., Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClearT:+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140 (6), 1364-1368 (2013).</li><li>Rinaldi, V. D., Hsieh, K., Munroe, R., Bolcun-Filas, E. M., Schimenti, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Inhibition+of+the+DNA+Damage+Checkpoint+Prevents+Radiation-Induced+Oocyte+Death.">Pharmacological Inhibition of the DNA Damage Checkpoint Prevents Radiation-Induced Oocyte Death.</a> Genetics. , (2017).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Obata, Y., Kono, T., Hatada, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis:+Maturation+of+mouse+fetal+germ+cells+in+vitro.">Oogenesis: Maturation of mouse fetal germ cells in vitro.</a> Nature. 418 (6897), 497 (2002).</li><li>Faire, 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=Follicle+dynamics+and+global+organization+in+the+intact+mouse+ovary.">Follicle dynamics and global organization in the intact mouse ovary.</a> Developmental Biology. 403 (1), 69-79 (2015).</li><li>Byrne, C., Hardman, M. 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Studien av däggdjur reproduktion kräver hjälp och kvantifiera specialiserade celler som inte rutinmässigt mottagliga för cellodling. Men är ex vivo kultur system effektiva på att upprätthålla äggstock och follikeln livskraft1,15. Under äggstock kultur kräver vävnaden en större yta för utbyte av näringsämnen genom diffusion. Därför 5 - dag-gammal mus äggstockarna är perfekta i storlek och form för orgelkultur. Detta protokoll var opt...

För att studera cellulära sammansättningen och morfologiska egenskaper av däggdjur äggstockar, forskare förlitar sig ofta på i vivo experiment följt av immunohistological färgning av paraffin inbäddade äggstockarna. Mer nyligen dock hela äggstocken orgelkultur har visat sig vara ett effektivt alternativ till studien äggstockarnas funktion1,2,3,4 eftersom tekniken kan kopplas till bättre visualisering och verktyg för kvantifiering. Traditionellt, analys av äggstockscancer morfologi är beroende av rekonstruktionen tredimensionella cystor arkitekturen från paraffin inbäddade seriell avsnitt, men, förutom att vara mödosam och tidskrävande, seriellt snittning paraffin inbäddade vävnad gör inte garanti ordentlig ombyggnad av orgeln, och sektioner ofta förloras eller mis beställde i processen.
Förutom de tekniska utmaningar i samband med seriell sektionering, finns det också variationer i de metoder som rutinmässigt används för att kvantifiera follikeln nummer per äggstock5,6. Det metodologiska variabilitet som för närvarande används försämrar meta-analys av äggreserven över studier5,7. Till exempel varierar follikeln nummer från olika forskningsartiklar 10-faldig eller mer mellan liknande utvecklingsmässiga åldrar inom en viss stam6. Dessa stora skillnader i rapporterade follikeln kvantifiering kan leda till förvirring och har hindrat cross-studien jämförelser. Experimentellt, traditionella metoder för follikeln kvantifiering från seriell avsnitt utförs genom att räkna folliklar av ett fördefinierat antal sektioner (t.ex. var femte, tionde, eller andra avsnitt). Variabilitet i follikeln räknar med detta synsätt uppstår inte bara från periodiciteten i vilka delar räknas men även från variationer i snittjocklek och teknisk erfarenhet inom generera serial avsnitten5,6. Förutom dess variationer är en annan nackdel med traditionell vävnad snittning att snittning av små äggstockar från unga djur är särskilt utmanande och starkt beroende av vävnad orientering8.
Protokollet nedan beskriver en rutinmässigt används äggstock kultur teknik1 men förbättrar kraftigt traditionella follikeln kvantifiering genom att ersätta fysiska snittning med vävnad clearing och optisk snittning med konfokalmikroskopi8 ,9. Rensa med vävnad nedsänkning (utan behovet av transkraniell perfusion eller elektrofores) i en urea - och sorbitol-baserade lösning (t.ex. ScaleS(0)10) visat sig vara kompatibla med immunfärgning och tillåtet för minskning av avfärgningstiden utan att kompromissa med djup Imaging. Andra rapporterade metoder (t.ex., ScaleA28,10, SeeDB11, ClearT12och ClearT212) är antingen mer tidsödande eller Tillåt inte djupgående optisk upplösning av provet. Optisk snittning är fördelaktigt eftersom det är mindre arbetsintensivt och upprätthåller de en orgel tredimensionella arkitektur7,8. En annan fördel med denna metod är att beredningen av proverna inte kräver kostsamma reagenser att rensa vävnaden och kan utföras med relativ lätthet.
Specifikt, det protokoll som beskrivs har optimerats för odlade mus äggstockarna på postnatal dag fem men har utförts på äggstockarna alltifrån postnatal dag 0 - 10. Metoden använder sig av en äggstock kultur system där vävnaden naturligt tillmäter membranet som är det odlade, underlätta orgel hantering och manipulation. Kultur systemet beskrivs kan användas för att upprätthålla explanterad äggstockarna för upp till 10 dagar och att bedöma hur olika experimentella förhållanden kan störa äggcellen överlevnad13. Rapporteringsgräns förfarandet utförs med icke-kommersiella bildbehandling paketet FIJI-ImageJ14 och kan utföras på de flesta persondatorer. Bilder som används för kvantifiering kan dessutom göras allmänt tillgängliga för forskarsamhället, vilket möjliggör framtida metaanalys.

<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

Forskning inom området för däggdjur reproduktionsbiologi innebär ofta utvärdera övergripande hälsa för äggstockar och testiklar. Specifikt, i kvinnor bedöms cystor fitness ofta av visualisera och kvantifiera folliklar och oocyter. Eftersom äggstockarna är en ogenomskinlig tredimensionella vävnad, kräver traditionella metoder mödosamt skivning vävnaden i många seriella sektioner för att visualisera cellerna i hela hela orgeln. Dessutom eftersom kvantifiering av denna metod innebär vanligtvis scoring endast en delmängd av avsnitten åtskilda av ungefärlig diameter en äggcell, är det benägna att felaktighet. Här beskrivs ett protokoll som istället utnyttjar hela orgel vävnad clearing och immunofluorescens färgning av mus äggstockarna att visualisera folliklar och oocyter. Jämfört med mer traditionella metoder, detta protokoll är fördelaktigt för att visualisera celler i äggstocken för många skäl: 1) äggstocken förblir intakt under hela provberedning och bearbetning. (2) små äggstockar, som är svåra att avsnitt, kan undersökas med lätthet; (3) cellulär kvantifiering uppnås lättare och korrekt; och 4) hela orgeln avbildas.

Detta protokoll innehåller 6 stora steg efter dissektion av äggstockarna, som beskrivs i figur 1. Figur 2 , Figur 3 , Figur 4 highlightar mest roman av detta protokoll, som omfattar optimering av vävnad clearing för äggstockar och hela vävnad äggcellen kvantifiering med FIJI-ImageJ. Figur 2...

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

Här presenterar vi ett protokoll för att kvantifiera folliklar i odlade äggstockarna av unga möss utan seriell sektionering. Använda hela orgel immunofluorescens och vävnad clearing, ersätts fysiska snittning med optisk snittning. Denna metod för provberedning och visualisering underhåller orgel integritet och underlättar automatiserade kvantifiering av specifika celler.

Hela berget immunofluorescens och follikeln kvantifiering av odlade mus äggstockarna

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

Kultur och Co-kultur av Mouse Äggstockar och folliklar

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

Isolera stamceller i hudens hårsäckar och epidermal keratinocyter från Dorsal mushud

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

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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

Organ kultur och Hela Mount immunofluorescensfärgning av Mus Wolffian kanaler

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

Visualisera Multiciliated celler i zebrafisk genom en kombinerad protokoll av hela Mount fluorescerande In Situ hybridisering och immunofluorescens

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

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Hela-mount Clearing och färgning av Arabidopsis blomma organ och 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

Kvantitativa hela-mount immunofluorescens analys av hjärt stamceller populationer i musembryon

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

Visualisera den noden och Notochordal plattan i Gastrulating musembryon som använder Scanning Electron Microscopy och hela Mount immunofluorescens

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

Analys av hjärtats kammare utveckling under musen embryogenes använder hela 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

Laserinfångning mikrodissektion av mus embryonala brosk och ben för gen uttrycks analys

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