We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

NOT: Tüm farenin Laboratuar Hayvan Hastalıkları Michigan Tıp Fakültesi Birimi Üniversitesi tarafından onaylanmış ve Hayvanları Kullanımı ve Bakımı Üniversite Komitesi kurallarına göre protokollerini kullanarak insanca ele alındı. 1. Tüm Organ Kültür Sistemi <ol><li> Medya ve kültür plakalarının hazırlanması <ol><li> Bir doku kültürü kaputu, stok şişe edilebilen BGJb ortamdan 5 ml kaldırmak ve Kalem / Strep 5 ml ekleyin. 5 mg / (Pen / Strep) ile edilebilen BGJb ortam 49 ml ml askorbik asit 1 ml ilave etmek suretiyle, 50 ml&#39;lik konik bir tüp içinde bir ortam çalışma stok hazırlayın. </li><li> Transwells her altında 1 ml ilave etmek suretiyle, bir 6 yuvalı kültür plakası hazırlayın. NOT: Tüm 6 transwells kullanılan değilseniz, daha sonra kullanmak üzere yeni bir steril 6 plaka ekstra transwells taşıyın. Üç adet 10 cm steril petri kutularının her birine edilebilen BGJb ortam çalışma 3 ml ilave edilir. </li></ol></li><li> Diseksiyon için Hazırlık <ol><li> 70% Etal içinde diseksiyon araçları batırınve metanol çalışırken temiz araçları tutmak için emin olun. </li><li> Büyük bir buz kovası ile diseksiyon alanı ayarlayın ve 6 kuyu kültür plaka ve buz üzerinde edilebilen BGJb medya ile 10 cm petri kapları yerleştirin. Diseksiyon ve kültür için bağırsakları hazırlarken mümkün olduğunca buz üzerinde çalışın. </li><li> Fetal bağırsakların toplama için servikal dislokasyon izofluoran (100 ul) önce euthanization için olan bir oda içinde erişkin dişi fareler anestezi. </li><li> Onların anneden fetusa hasat ettikten sonra, her fetusun dikkatli grafiği 13 evreleme Theiler göre düzenlerler. Rutin bir tek çöp içinde gözlenen gelişimsel aşamada kadar 24 saat varyasyonları olarak her fetusun yaşını belirlemek için post-koitus günlerde güvenmeyin. </li></ol></li><li> Fetal bağırsak diseksiyonu <ol><li> Bağırsakların kaldırılması için önce, başı kesilerek fetüsleri Euthanize. </li><li> Pla sonra 1x Dulbecco Fosfat Tamponlu Salin (DPBS) her bağırsak inceleyin veedilebilen BGJb ortamı çalışan bir 10 cm&#39;lik bir petri kabı içine ce. </li><li> Dikkatlice mide, dalak, mide ve oniki parmak bağırsağı üst elde edilen pankreasa, kaldırın. </li><li> Yavaşça doğruldu bağırsak izin mümkün ve yalnızca yeterli bağlantılarını ayıran olduğunca bağlı omentum bırakmak için özen omentum ayırın. Not: yavaşça lümen içeriği bağırsak yoluyla akması ve kesik uçlarından çıkarılmasına imkan vermek için 3 eşit bölümlere ayırmak bağırsak bağırsak E14.5 daha büyük veya daha fazla olanlar, 24 saat daha kültive edilir için. Bununla birlikte, kültürlerin E13.5 önce, serozayı uygun bağırsak uzunluğu boyunca büyümeye izin bağırsak 3 kısımları ayıran önce kültürlenmesi kadar 24 saat sonra başladı bekleyin. </li><li> Kültür plakası (1.1.2) transwells üzerine bağırsak parçaları aktarmak için bir ağız pipeti kullanılır. </li><li> Onlar transwel genelinde düz böylece gerektiği gibi yavaşça bağırsakları yeniden konumlandırmakl. NOT: bağırsaklar tüp yoluyla lümen içeriğini hareket başladıktan sonra, bağırsakta herhangi bir karışıklığı bağırsağa bir yedekleme ve hasara neden olur. </li></ol></li><li> Kültür ve tedavi <ol><li> , Transwell altından medyayı çıkarın transwell altında (ilave rekombinant proteinlerin veya farmakolojik inhibitörleri ile medya çalışma) tedavi medya 700 ul ekleyin. </li><li> Bağırsakların üstüne tedavi medya damla damla 300 ul ekleyin ve transwell yoluyla bekletin. Gerekirse bağırsakları yerleştirin. </li><li> Tedavi reaktif yarı ömrüne bağlı olarak en az günde bir kere ya da daha çok muamele ortam değiştirin. </li></ol></li><li> Tedavi reaktif lokalize bir protein kaynağı batırılmış agaroz taneleri hazırlanması <ol><li> Kendi depolama kabı agaroz boncuklar tekrar ve 1.5 ml mikrofüj tüpüne agaroz boncuklarının 100 ul transfer. </li><li> Steril 1x Ph borunun geri kalan hacim dolumunaosphate-tamponlu salin (PBS) ile yıkamak üzere, taneler üzerine karıştırın. Birkaç dakika boncuklar dibe ve daha sonra tanelerin üzerine PBS kapalı Pipeti izin vermek üzere askıya mikrofüj tüpü yerleştirin. Steril 1x PBS ile mikrofuge&#39;ye tüp dolum ve iki kez daha yıkama işlemini tekrarlayın. </li><li> Iki kez tedavi için istenen konsantrasyona ve ilgi konusu proteini hazırlayın. </li><li> 2x Protein stoklar 5 ul ile yıkanmış agaroz boncuk 5 ul karıştırın ve yavaşça protein boncuk çalkalayın. Yavaşça, 10-15 dakika daha karıştırıldı, 1 saat boyunca buz üzerinde tüp yerleştirin. </li><li> Bağırsakların boncuk yerleştirmeden önce steril 1x PBS kısaca boncuk durulayın. </li></ol></li><li> Agaroz boncukları Yerleştirme <ol><li> Yavaşça çekilmiş kılcal iğneli bir ağız pipeti kullanılarak bağırsakların üst agaroz boncuklar yerleştirin. Kaba işleme bağırsaklara zarar ve yaralı bölgede villus gelişimini önleyecek şekilde aşırı ihtiyatla boncuk yerleştirin. </li><li> Bağırsak peristaltik hareketleri ve kültür zamanla bağırsakların inecektir yerleştirilen boncuklar yaklaşık yarısını geçirecek beri analiz için gerekli iki katı kadar boncuk yerleştirin. </li></ol></li><li> Kültürlü bağırsakların Fixation <ol><li> Dikkatle transwell alttan tedavi ortamı çıkarın ve 3 dakika boyunca havada kurumaya bağırsakları izin verir. </li><li> Daha sonra tespit süresinin geri kalan kısmı için sabitleyici 2 ml bağırsağın üst kapağı, 2 dakika boyunca transwell altında fiksatif 1 ml ilave edilir. 4 ° C&#39;de ya da oda sıcaklığında 2 saat süresince% 4 paraformaldehit O / N sabitleyin. </li></ol></li></ol> Sabit Bağırsaklar 2. Görüntüleme <ol><li> Wholemount görüntüleme <ol><li> DPBS 1x 100 ul, bir lam üzerine (iç floresan ile) bütün bağırsak yerleştirin. Yavaşça bağırsaklara düzenlemek için forseps kullanın. </li><li> Mo doku yapmak için dikkatli bir şekilde% 70 gliserol, 100 ul DPBS çıkarın ve hemen eklemek için bir laboratuar doku kullanınşeffaf yeniden ve görüntüleme olası derinliğini artırır. Not: bağırsağın başka penetrasyon isteniyorsa yerine% 70 gliserol içinde bağırsak Sabitleme, 10-30 dakika boyunca Odak sonuçları çözeltisinin bir kaç damla bağırsak ıslatın. Odak Temizle çözümü kapalı pipetlemeyin Mount Clear bağırsak monte edin. </li><li> Modelleme kil içine lamel dört köşesinden her batırın ve bağırsak düzleştirme lamel tutmak için slayt ve kapak arasında ayırıcı olarak kullanmak için kil küçük bir miktar pick up. </li><li> Bir pamuklu çubukla uygulanan erimiş valap ile slayt üzerine lamel mühür. </li><li> Görüntü ya bir dik veya ters konfokal mikroskop bağırsaklar. Daha fazla ayrıntı için tartışma bakınız. </li></ol></li><li> (Bağırsak yapıların enine kesit görünümü için) sabit bağırsak vibratome kesit <ol><li> Küçük plastik kalıplar% 7 agaroz katıştırmak için bağırsak (10 x 10 x 15 mm), izin agaroz bloklarsoğumaya ve katılaşmaya. </li><li> Anlık yapıştırıcı ile Vibratome toplama aşamasına plastik kalıp ve tutkal agaroz bloklardan agaroz blokları çıkarın. </li><li> Buz gibi soğuk 1x DPBS toplama sahne doldurun. </li><li> 100-150 um arasında bir kalınlıkta kesitler halinde kesilmiştir. Kalın kesitler kesiti içine derin görselleştirmek için (bölüm 2.1.2 açıklanan) takas çözümleri kullanın. </li><li> 4 ° C de depolama için, bir 6-yuvalı plaka içinde bir kuyu içine toplama aşamasından vibratome bölümleri dökün. </li></ol></li><li> Sabit, Vibratome kesitli dokuların İmmünofloresan boyanması <ol><li> 1x DPBS ile bir 24-yuvalı plaka içine, göz başına 5-12 vibratome bölümleri yerleştirin. </li><li> 1x DPBS çıkarın ve oyuk başına geçirgenleştirme çözeltisi 500 ul ilave edin. Yavaşça oda sıcaklığında 25 dakika boyunca sallayın örnekleri. </li><li> Geçirgenleştirmeden sonra, 1x PBS ile numuneleri 3 kere yıkayın. </li><li> Bloke etme çözeltisi 500 ul içinde en az 30 dakika boyunca numune bloke eder. </li><li> Engelleme, e sonraçözümü engelleme seyreltilmiş primer antikor 500 ul engelleme çözüm Xchange ve 4 ° CO / N de örnekleri tutmak. NOT: Dondurulmuş ve parafin bölümlerde işe birincil antikor dilüsyonları genellikle Ancak genellikle bu protokolü (örneğin, BrdU) ile iyi çalışmaz nükleer antijen alımı gerektiren antikorlar, çok Vibratome bölümlerde iyi çalışır. </li><li> Ertesi gün, bloke etme çözeltisi ile 15 dakika boyunca numuneleri her 3 kez yıkayın. </li><li> Yıkamadan sonra, bloke etme çözeltisi içinde seyreltilmiş sekonder antikor, 500 ul de geçerlidir. Oda sıcaklığında 2 saat 45 dakika boyunca örnekleri ışıktan korumak ve kaya florofor için alüminyum folyo ile 24 oyuklu plaka sarın. </li><li> 15 dakika her biri için 1x PBS ile numuneleri 3 kez yıkayın ve Bölüm 2.4 &#39;de açıklandığı gibi slaytlar üzerinde vibratome bölümleri monte edin. </li></ol></li><li> Vibratome bölümleri Montaj <ol><li> Vibratome çift sti ince dilim keserek montaj için slaytlar hazırlayınck bant ve slaytlar uzun kenarları boyunca onları yerleştirerek. </li><li> Dikkatle 1x PBS 100 ul bir damla slaytlar üzerinde çift kaset sopa arası 5-10 vibratome bölümleri yerleştirin. </li><li> Tüm bölümlerini açın ve slaytta tek bir tabaka içine yayıldı. </li><li> Düz oturan bir coverslip engelleyecek herhangi bir aşırı agarozun veya düzensiz parçalarını çıkarın. </li><li> Dikkatle Vibratome bölümler etrafında aşırı 1x PBS kadar emmek için bir laboratuar doku kullanın. </li><li> Vibratome bölümlerin üstüne monte sulu ortam bir damla ekleme ve sonra da lamel. </li><li> Valap bir pamuklu çubukla uygulanan eritilmiş slaytlar üzerine lamelleri Seal. </li><li> Görüntü ya bir dik veya ters konfokal mikroskop vibratome bölümleri. Bir görüntüleme sisteminin seçimi konusunda ayrıntılı bilgi için tartışmaya bakınız. </li></ol></li></ol> Tüm Bağırsaklar 3. Canlı Görüntüleme Bağırsaklar fetuse hasatBağlı omentum sağlam sol s ile E12.5 sonra, başarıyla yukarıdaki sistemini kullanarak kültür içinde villus gelişir. Bu nedenle, bu ex vivo sistem zamanla morfolojilerinden bir üç boyutlu görünüm için yeniden olabilir gelişen bağırsak canlı z-bölüm görüntüler yakalama izin verir. <ol><li> Canlı görüntüleme ayarlama <ol><li> Ince örgülü elek için tek bir polikarbonat membran bağlı anında tutkal. Bu dik konfokal mikroskop daldırma lens aşağıda yerinde bağırsak tutmak için bir destek iskele sağlar. Canlı görüntüleme mikroskoplar seçimi hakkında ayrıntılı bilgi için tartışma bölümüne bakınız. </li><li> Merkezinde iyi bir kültür tepsisi içine elek yerleştirin. </li><li> Polikarbonat membran üzerine disseke bağırsak yerleştirin ve her iki ucunda anlık tutkal bir damla ekleyin. Bağırsak tutturmak için yalnızca yeterli tutkal kullanın, ama ceket o! </li><li> Polikarbonat altında fenol kırmızısının kültür ortamı ücretsiz eklentiMerkezi de kültür plakasında membran. </li><li> Nem temin etmek üzere kültür plakasının dış kenarı için su ilave edilir. </li><li> Görüntüleme sırasında bağırsak hareketini kontrol etmek için ortam 3 ml 1 x PBS&#39;de ksilazin 5 çözeltisi: fetal barsak kültürde peristaltik kasılma benzeri dalgalar maruz kaldığından, 1 100 ul ilave edin. Bu dozaj villus gelişimini ödün vermeden yaklaşık 8-10 saat bağırsak hareketlerini kontrol edecek. </li><li> Bağırsağın bir enine görünüm için, 3-4% agaroz çözeltisi içinde canlı bağırsakları gömmek ve bir vibratom kalın kesitler (150-500 mikron) kesilir. Kesilmiş olarak, bağırsak sertliğe sahip agaroz sertliğini maç ve ampirik olarak bir gelişme aşamasındaki belirlenir. Genellikle,% 3 agaroz çözüm E14.5 daha genç bağırsaklar için iyi çalışır ve% 4 çözelti bağırsaklar E15-E16 için iyi çalışır. </li><li> (Kısım 3.1.1 gibi) gözenekli bir elek tutturulmuş bir polikarbonat membran vibratome bölümleri ve TutkalKültür olarak Bölüm 3.1.2-3.1.6 tarif edilmiştir. </li><li> Ayarlanabilir bir tablo ile dik bir stand üzerine bir iki-foton konfokal floresan mikroskop (690-1,040 nm arasında uyarma lazer) kullanılarak canlı görüntüleme Davranış. </li><li> İki foton lazer GFP sinyalleri için en iyi çözünürlük ile derin penetrasyon sağlar nm 900 ayarlı. Görüntülenir iken bu dokulara zarar azaltır. Buna ek olarak, mümkün olan en düşük emisyon için lazerin gücünü ayarlayıcı, doku hasarı azaltır. Tipik olarak, GFP iyi bir uyarma elde iki foton lazer% 12 güç kullanmak. </li></ol></li></ol>

<ol>
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The authors have no financial conflicts to disclose.

Dinamik yapısı ve gelişen bağırsak karmaşık doku etkileşimleri bu morfogenetik olayların tam bir takdir 3D görselleştirme gerektirir. Görüntüleme Gelişen teknolojiye, yetenek / gelişmekte iyileştirilmesi ve onunla, organogenezis sırasında mekansal iletişim ve etkileşim anlayışı oldukça gelişmiş edilir detaylı olarak villus morfogenetiğine incelemek. Bütün bağırsakları kültürü için alternatif yöntemleri de test edilmiştir, ancak Transwell sistem uzun k?...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="1308">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fine dissecting forceps&#160;</td>
			<td style="width:223px;">Fine Science Tools</td>
			<td style="width:133px;">&#160;11254-20</td>
			<td style="width:444px;">2 pairs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x sterile Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Gibco&#160;</td>
			<td>14040-133</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plates</td>
			<td>Costar&#160;</td>
			<td>3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 x 15 mm petri dishes</td>
			<td>Falcon</td>
			<td>&#160;451007</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td>
			<td>Corning Costar&#160;</td>
			<td>3428</td>
			<td style="width:444px;">6 inserts per plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BGJb media</td>
			<td>Invitrogen&#160;</td>
			<td>12591-038</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td>
			<td>Gibco</td>
			<td>&#160;15140</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ascorbic Acid</td>
			<td>Sigma&#160;</td>
			<td>A0278</td>
			<td style="width:444px;">make 5 mg/ml stock, filter, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td>
			<td>Fisher</td>
			<td style="width:133px;">&#160;21-180-10</td>
			<td style="width:444px;">remove the aspirator assembly and replace it with a 1000 &#181;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 inch glass pasteur pipets</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose beads</td>
			<td>BioRad&#160;</td>
			<td>153-7301</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary Tubes</td>
			<td>World Precision Instruments&#160;</td>
			<td>TW100F-4</td>
			<td style="width:444px;">pull to needles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td style="width:444px;">made in 1 x PBS, pH to 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live Imaging Materials</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture plates</td>
			<td>Falcon&#160;</td>
			<td>353037</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine mesh stainless steel screen</td>
			<td>purchase at hardware store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Polycarbonate membranes</td>
			<td>Thomas scientific&#160;</td>
			<td>4663H25</td>
			<td style="width:444px;">alternatively, cut Corning Costar 3428 membranes off of transwell supports</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Instant glue</td>
			<td>purchase at hardware store</td>
			<td style="width:133px;">gel based preferrably</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 x 10 mm plates</td>
			<td>Falcon&#160;</td>
			<td style="width:133px;">351008</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7% agarose</td>
			<td>Sigma</td>
			<td>&#160;A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minutien pins</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:133px;">26002-20</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol red free media (DMEM)</td>
			<td>Gibco&#160;</td>
			<td style="width:133px;">21063-029</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (100 mg/ml)</td>
			<td>AnaSed&#160;</td>
			<td style="width:133px;">139-236</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Matrigel</td>
			<td>BD</td>
			<td style="width:133px;">356231</td>
			<td style="width:444px;">basement membrane matrix, growth factor reduced, phenol red-free</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3-4% agarose</td>
			<td>Sigma&#160;</td>
			<td>A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging of fixed intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vaseline</td>
			<td style="width:223px;">purchase at pharmacy</td>
			<td></td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lanolin</td>
			<td style="width:223px;">Sigma&#160;</td>
			<td>L7387</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:223px;">Surgipath</td>
			<td>39601006</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% glycerol in 1 x PBS</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Focus clear and Mount Clear</td>
			<td>CelExplorer Labs Co.</td>
			<td>&#160;F101-KIT</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modeling clay</td>
			<td>purchase at art supply store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double stick tape</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cotton applicator swabs</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic molds, 10mm x 10mm x 5 mm)</td>
			<td>Tissue Tek&#160;</td>
			<td>4565</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">slides</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coverslips</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lab wipe</td>
			<td>Kimberly Clark</td>
			<td>34155</td>
			<td style="width:444px;">lint free delicate task wipe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Theiler staging chart&#160;</td>
			<td></td>
			<td></td>
			<td style="width:444px;"><a href="http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf">http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica SP5X confocal microscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMI 6000 stand&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Aqueous mounting medium (Prolong Gold)</td>
			<td>Molecular Probes&#160;</td>
			<td>P36930</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for Immunofluorescence staining of fixed, vibratome sectioned intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plate</td>
			<td>Costar</td>
			<td>&#160;3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td style="width:444px;">used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Goat serum</td>
			<td></td>
			<td></td>
			<td style="width:444px;">used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tween20&#160;</td>
			<td>Sigma</td>
			<td>&#160;P9416</td>
			<td style="width:444px;"></td>
		</tr>
	</tbody>
</table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

Fetal bağırsakta en morfogenetik süreçler gelişim aşamaları üzerinde değişikliklerin anlık sağlayarak, sabit dokuların ince kesitlerinin olayla edilmiştir. İnce Seri kesitlerde üç boyutlu bilgiler, çünkü mükemmel seri kesitler yeniden ve seri kesitler üzerinde dokusunun uygun yönünü sürdürmenin zorluğu yorumlamak zor olabilir. Tarafından Grosse ve ark. Son bulgular, 2011 bağırsak 1 gelişmekte villuslarda morfogenetiğine anlamada üç boyutlu bilgilerin önemini vurgulamak. Tek başına etiketli bağırsak hücreleri üç boyutlu yeniden yapılandırma bağırsak epitel hücrelerinin çoğunluğu her iki en üst ve taban yüzeylerine temas gösterdi. Ayrıca, epitel apikal yüzeyinde aktin hücre iskeletinin üç boyutlu rekonstrüksiyon bağırsak lümen sürekli olduğunu ve ikincil lümen s bir eserdir olduğunu gösterdiectioning. Bu iki nokta, bağırsak epitelinde interkinetic nükleer göç gösteri ile birlikte, bir yalancı epiteli olarak gelişen bağırsak epiteli tanımlanmış ve önceden 1 düşünce olarak tabakalı değil. Epitel üç boyutlu görebilme bu noktayı gösteren ve fetal bağırsakta epitel morfogenetiğine yeniden tanımlıyor ufuk açıcı oldu. Çoklu foton görüntüleme teknolojisi ve üç boyutlu rekonstrüksiyon yazılım evrimi ile, yeteneği hızla iyileştirilmesi organ geliştirmeyi, sağlam görselleştirmek için. İki foton uyarma yüksek çözünürlüklü dokulara daha az zarar penetrasyon derin verir. İki foton görüntüleme ve Walton vd. Tüm fetal fare bağırsak 3D rekonstrüksiyon 2012 villus akıbet 2 deseni tanımlamak için yardımcı oldu. Burada izin vermek için bu kültür sisteminin villus ve uzantıları ex vivo gelişimi sağlayan bir bütün organ kültür sistemi açıklanmaktadırbağırsaklar üç boyutlu gelişim sırasında görüntülenmiş olması.

Bu sistem farmakolojik reaktifler veya rekombinant proteinleri ile sinyalizasyon manipülasyon sağlayan fetal Bağırsakların tüm eksplantlarının Kültür, bağırsak gelişimini koordine sinyal moleküllerinin konumu, dağılımı ve süresi analizler için izin verir. (Walton ve ark yeniden Şekil 1A., 2012) Transwell kültür sistemi 2 mümkün doku üzerine ilaç veya proteini batırılmış agaroz boncuklar yerleştirmek ve böylece lokal sinyal kaynaklarının bir...

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

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.

Için Fare Fetal Tüm Bağırsak Kültür Sistemi<em&gt; Ex vivo</em&gt; Sinyal facebook ve villus Kalkınma Üç boyutlu Canlı Görüntüleme Manipülasyon

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

mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Research

JoVE Journal

Biology

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

14.8K Görüntüleme.  University of Michigan. 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. 

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

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biyoloji

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Bir Eski-ovo</emGörüntüleme ve Mikrocerrahi Uygulamaları için uygundur> Tavuk Embriyo Kültürü Sistemi

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Rotator-tipi Şişe Kültür Sistemi kullanılarak Kemirgen Tüm Embriyo Kültürü Yöntemi

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Için bir sistem<em&gt; Ex vivo</emEmbriyonik Pankreas&gt; ekimi

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Serumsuz Tüm Embriyo Kültür Sistemde Fare Embriyonik Development

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Fare Embriyonik Cilt ve Melanoblast Göç Canlı görüntüleme ex vivo Kültür

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Bir<em&gt; Ex vivo</em&gt; Kültür Sistemi Tiroid Geliştirme Çalışması

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Üç boyutlu Hidrojeller içinde Görüntüleme FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...