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

Vi vil gerne takke medlemmerne af gynækologi onkologi gruppen for kritisk læsning af dette manuskript. Dette arbejde er delvist støttet af midler fra National Health og Medical Research Council, Australian Research Council, og Cancer Institute NSW (PST). MK er en modtager af University of Newcastle Postgraduate Research Fellowship.

Animal pleje og eksperimentelle procedurer blev udført i overensstemmelse med retningslinjerne i Animal Care og etiske komité fra University of Newcastle og bekræftet til Forskning lov om New South Wales Animal, New South Wales Animal Research forordning, og den australske kode for pleje og brug af dyr til videnskabelige formål. Alle de procedurer, der gennemføres på mus blev godkendt af Animal Care og etiske komité fra University of Newcastle. 1. Time Parring <ol><li> Pair 6 - 8 ug...

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	<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>

Den organkultur system har mange fordele frem for den traditionelle cellekultursystem. Dette system bevarer de oprindelige strukturelle relationer og interaktioner mellem forskellige celletyper. Det efterligner in vivo-systemer og giver mere præcise oplysninger end cellekultur studier, hvor den niche faktor er fraværende. Anvendelsen af ​​organsystemer dyrkningssystemer endda have fordele ved at benytte hele dyret. Disse omfatter de højere omkostninger ved at bruge hele dyret, nem pleje og vedligeholdels...

Den mandlige reproduktive system består primært af testis, stedet for kimcelle udvikling og differentiering og et komplekst gangsystemet, der kræves for modningen, transport og opbevaring af sædceller. Epididymis er en rørformet organ, der forbinder testis med sædlederen og hovedsageligt involveret i post-testikelkræft udvikling og modning af kønsceller 1. De stærkt coiled voksne mus epididymis udvikler sig fra en enkel og lige precursor rør, Wolffian kanal (WD) 1. Komplekset og oprullede struktur epididymis er afgørende for sædcellerne at erhverve evnen til at befrugte kvindelige kønsceller 2. Hvordan sådan en vigtig organ for mandlig fertilitet udvikler og kommer i form er ikke godt forstået. Forskellige signalveje er blevet vist at være involveret i udviklingen af WD såsom Wnt signalvejen 3,4 og androgenet signalvejen 5. Vores seneste arbejde har etableret kravet om balbalanceret Wnt signalering til WD oprulning under prænatal udvikling 4. Imidlertid er yderligere undersøgelser nødvendige for at forstå de mekanismer, der er involveret i postnatal epididymalt coiling, cellulær differentiering, og celle-til-celle kommunikation i epididymale epitel og med interstitielle celler. Genetisk modificerede musemodeller har vist sig at være et effektivt redskab til identifikation / validering af molekylære mekanismer bag udvikling og sygdom af forskellige organsystemer 6. Trods dens omfattende brug, er der væsentlige begrænsninger af genetisk modificerede musemodeller, herunder uforudsete fænotype af målrettede mus mutanter med hensyn til formodede genfunktion, og ingen fænotype i nulmutanter i nogle modeller, indflydelse af genetiske og miljømæssige faktorer, arbejdskrævende og tidskrævende proces med at udvikle musemodeller. Derfor fortolkningen af ​​betydningen af ​​resultaterne kun fra genetisk modicerede musemodeller er ikke altid ligetil 6. Disse begrænsninger kan overvindes ved hjælp af in vitro-orgel kultur system, der giver os fleksibilitet til at arbejde med flere signalveje i realtid i kontrollerede dyrkningsbetingelser. Organkultur system er medvirkende til at forstå fysiologi og patologi hele organer 7. Endvidere billeddannelse af hele organer farvet med fluoroformærkede antistoffer kan vi visualisere markører af interesse i en tredimensionel kontekst, som den eksisterer in vivo, hvorved der tilvejebringes en bedre forståelse af organets form, struktur og funktion 8. Her har vi beskrevet de metoder til isolering af muse embryonale gonadale kamme, in vitro kultur og hele mount immunfluorescens billeddannelse af WDS, der kan anvendes til at besvare en række spørgsmål om morfogenese af rørformede organer såsom WDS. I denne protokol, viisolerede muse embryonale urogenitale kamme fra 15,5 dage efter coitum (dpc) gravide dæmninger og dyrket dem i 3 dage efterfulgt af immunfarvning for en epitelcelle markør (cytokeratin 8, Ck8), en celle proliferation markør (phospho-histon 3, PH3) og aktiv βcatenin.

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			<td height="42" style="height:42px;width:247px;">24 well plates</td>
			<td style="width:195px;">Corning, USA</td>
			<td style="width:136px;">3524</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td style="width:195px;">GE Healthcare Life Sciences, USA</td>
			<td style="width:136px;">SH30031.02</td>
			<td style="width:191px;">can be purchased from other vendors</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">D8437</td>
			<td style="width:191px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:195px;">Interpath,&#160; Australia</td>
			<td style="width:136px;">SH30034.02</td>
			<td style="width:191px;">10% FBS used in culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">L-Glutamine</td>
			<td style="width:195px;">GE Healthcare Life Sciences, USA</td>
			<td style="width:136px;">SH30034.01</td>
			<td style="width:191px;">1% concentration used</td>
		</tr>
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			<td height="62" style="height:62px;width:247px;">Penicillin-Streptomycin</td>
			<td style="width:195px;">Gibco, USA</td>
			<td style="width:136px;">15070-063</td>
			<td style="width:191px;">1% concentration used</td>
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			<td height="21" style="height:21px;width:247px;">Whatman Nuclepore Polycarbonate Track-Etch&#160;</td>
			<td style="width:195px;">Whatman, USA</td>
			<td style="width:136px;">110409</td>
			<td style="width:191px;">Membrane (0.8 mm)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">IWR1</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">10161-5mg</td>
			<td style="width:191px;">100 &#181;M concentration used</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">Paraformaldehyde (PFA)</td>
			<td style="width:195px;">Electron Microscopy Sciences, USA</td>
			<td style="width:136px;">15710</td>
			<td style="width:191px;">16% stock, 4% in PBS for use. TOXIC - wear gloves and cannot 
			be disposed of in the sink</td>
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			<td height="42" style="height:42px;width:247px;">Ethanol</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">214-20L PL</td>
			<td style="width:191px;">Absolute, make 25, 50 and 75% with milliQ water.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Tween-20</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">2509-500ml</td>
			<td style="width:191px;">Very viscous, careful while dispensing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Triton X-100</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">T8787-50ml</td>
			<td style="width:191px;">Very viscous, careful while dispensing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Di-Sodium Hydrogen Phosphate</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">621-500mg</td>
			<td style="width:191px;">can be purchased from other vendors</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Sodium Di-hydrogen Phosphate</td>
			<td style="width:195px;">Ajax Finechem, USA</td>
			<td style="width:136px;">4745-500g</td>
			<td style="width:191px;">can be purchased from other vendors</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Sodium Chloride</td>
			<td style="width:195px;">Thermo Scientific Fisher, USA</td>
			<td style="width:136px;">465-500g</td>
			<td style="width:191px;">can be purchased from other vendors</td>
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		<tr height="83">
			<td height="83" style="height:83px;width:247px;">Cytokeratin8/Troma I</td>
			<td style="width:195px;">Developmental Studies Hybridoma Bank, USA (DHSB)</td>
			<td style="width:136px;">TROMA-I</td>
			<td style="width:191px;">1:250 in blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">active &#946;catenin&#160;</td>
			<td style="width:195px;">Cell Signalling Technology,&#160; USA</td>
			<td style="width:136px;">D13A1</td>
			<td style="width:191px;">1:200 in blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;"></td>
			<td style="width:195px;"></td>
			<td style="width:136px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">Phospho-Histone3</td>
			<td style="width:195px;">Millipore, MA, USA</td>
			<td style="width:136px;">06-570</td>
			<td style="width:191px;">1:200 in blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 Goat anti-rabbit IgG</td>
			<td style="width:195px;">Jackson ImmunoResearchLabs,&#160; USA</td>
			<td style="width:136px;">111-545-047</td>
			<td style="width:191px;">1:250 in blocking buffer</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:247px;">Alexa594 Goat anti-Rat IgG</td>
			<td style="width:195px;">Jackson ImmunoResearchLabs,&#160; USA</td>
			<td style="width:136px;">112-585-072</td>
			<td style="width:191px;">1:250 in blocking buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Glycerol</td>
			<td style="width:195px;">Thermo Fisher Scientific, USA</td>
			<td style="width:136px;">242-500ml</td>
			<td style="width:191px;">can be purchased from other vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">n-Propyl galate</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">2370</td>
			<td>-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI)</td>
			<td style="width:195px;">Sigma, USA</td>
			<td style="width:136px;">D9564-10mg</td>
			<td style="width:191px;">Use to stain nucleic acids (DNA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:195px;">Thermo Fisher Scientific, USA</td>
			<td style="width:136px;">2225-500ml</td>
			<td style="width:191px;">Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cover Slips (24x50 mm)</td>
			<td style="width:195px;">Lomb Scientific</td>
			<td style="width:136px;">CS24501GP</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

organ culture and whole mount immunofluorescence staining of mouse wolffian ducts

Tubal morfogenese er en grundlæggende forudsætning for udviklingen af ​​de fleste pattedyr organer, herunder den mandlige reproduktive system. De epididymis, en integreret del af den mandlige reproduktive tarmkanalen, er ansvarlig for opbevaring af sæd, modning og transport. Den voksne epididymis er en meget coilrør der udvikler sig fra en enkel og lige embryonale precursor kendt som Wolffian kanal (WD). Korrekt oprulning af epididymis er afgørende for mandlige fertilitet, som sædceller i testiklerne ikke kan befrugte en oocyt. Men den mekanisme, der epididymis udvikling og oprulning er fortsat uklart, delvist på grund af manglen af ​​hele organkultur og billeddannende metoder. I denne undersøgelse beskriver vi et in vitro kultursystem og hele mount immunofluorescens protokol bedre at visualisere processen med WD oprulningen og udvikling, som også kan anvendes til at studere andre rørformede organer.

Under udvikling, WD foretages væsentlige ændringer, hvor en enkel og lige rør er omdannet til en meget kompleks og sammenrullet kanal. Anvendelse af fremgangsmåder beskrevet ovenfor, WDS undergår en lignende transformation i dyrkningsbetingelser. Her har vi vist resultaterne fra WDS dyrket i 3 d (figur 3). At dissekere molekylære mekanismer involveret i WD morfogenese, kan dyrkningsmediet suppleres med inhibitorer og aktivatorer rettet mod forskellige signalveje. Figur 3C viser ru...

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

Vi præsenterer en fremgangsmåde til isolering og dyrkning af muse Wolffian kanalen (WD). Vi viser også en detaljeret procedure for hele mount immunfarvning af dyrkede / frisk isolerede WDS med fluorescens mærkede antistoffer. Tilsammen udgør disse teknikker gør det muligt at studere WD udvikling, opvikling, og differentiering.

Organ Kultur og Whole Mount Immunofluorescens Farvning af Mouse Wolffian Kanaler

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

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