Bu eser Deutsche Forschungsgemeinschaft SFB688 (için A.G.) tarafından desteklenmiştir.

Fare üreme Regierung Unterfranken tarafından kabul edildi ve tüm analizler tüm Alman ve Avrupa Birliği yasalara ve yönetmeliklere bakım ve laboratuvar hayvanlarının kullanımı ile ilgili sıkı doğrultusunda gerçekleştirilmiştir.
1. Microtiter kaplama α4β1 Integrin ile Eski Utero Allantois Explant kültür için tabaklar
<ol><li>200 µg/mL steril fosfat tamponlu tuz çözeltisi (PBS), fare α4β1 liyofilize integrin sulandırmak. Önceden ısıtılmış (37 ° C) PBS 10 µg/mL nihai bir konsantrasyon oranında seyreltin. Pipet 20 µL/kuyu kuyu microtiter plaka (1 allantois/de) gerekli sayıda seyreltilmiş α4β1 integrin çözümde. Not: microtiter plaka kuyu kaplama hava kabarcıklarının oluşumunu önlemek.</li>
	<li>37 ° C'de 60 dk microtiter plakaları bir oksijen doku kültürü kuluçka makinesine yerleştirerek kuluçkaya.</li>
	<li>Dikkatle süpernatant bir pipet ile Aspire edin. Pipet ucu iyi alt dokunmadan önlemek için plaka eğim ve yumuşak emici kullanın. İki kez Önceden ısıtılmış (37 ° C) kültür ortamının gibi Dulbecco'nın modifiye kartal orta (DMEM) 50 µL ekleyerek Wells yıkama ve çamaşır orta yumuşak emici tarafından kaldırın.</li>
	<li>Kritik adım: ücretsiz siteleri 50 µL/kuyu %0,1 (w/v) sığır serum albumin (BSA) ekleyerek bağlama blok / DMEM ve oksijen doku kültürü kuluçka makinesine microtiter plakaları yerleştirerek ≥ 37 ° C'de 30 dk için kuluçkaya. Not: Allantois explants varlığında serum çeşitli yüzeyler ekleyebilirsiniz. Bu nedenle non-spesifik bağlayıcı siteleri engellemek için şarttır, kaplama sonra kuyu integrin ile disseke allantois eklemeden önce serum içeren kültür explant orta (bkz. Adım 4.4).</li>
	<li>Kaplamalı wells adım 4.4 gerekinceye kadar 37 ° C'de engelleme çözümde tutun.</li>
</ol>2. rahim diseksiyon Dpc 8, hamile bir fareden
Not: ağırlık kazanç ayrımcılık15tarafından yanlış pozitif gebelik oranları azaltmak.
<ol><li>Fare tarafından servikal çıkığı kurban. Karın bölgesi kürkle % 70 (v/v) etanol ile püskürtülerek dezenfekte. Not: fareler işlerken eldiven. Ötenazi anestezi olmadan servikal çıkığı tarafından allantois eki ile girişime neden olabilir allantois kimyasal kirlenme engeller.</li>
	<li>Karın deri makasla orta hat küçük bir kesi yaparak dosyayı açtı. Cilt kesi altında ve üstünde tutmak için eldivenli parmak kullanmak ve cilt göğüs ve fare kuyruğu doğru ayır.</li>
	<li>Karın zarını Forseps ile kavramak. Diğer el ile yapmak bir V şeklinde kesi gelen ön arka doğru makas kullanarak ve karın boşluğu açıklamakla doku taşıyın. Forseps bağırsak rahim erişim kazanmak için bir kenara itmek için kullanırız.</li>
	<li>İki rahim korna bulun. Forseps ile serviks (Kaudal kesim) tutun ve ligament iyi makasla kesme. Sonra her iki rahim boynuzları makasla oviducts kesilmesinin tarafından bağlantısını kesin.</li>
	<li>Forseps oda sıcaklığında (RT) PBS içeren bir steril 10 cm çanak içine steril, rahim aktarmak için kullanın. Yağ dokusu, damarları ve sinirleri çevreleyen güzel forseps ve/veya makas (şekil 1A) ile rahim boynuzları kaldırın.</li>
</ol>3. embriyo diseksiyon
Not: bir 8:1 zum mesafesi ile donatılmış bir stereomicroscope altında aşağıdaki adımları gerçekleştirin.
<ol><li>Embriyo implantasyonu siteler arasında keserek makas ile ayırın. Kas rahim astarı rahim tomurcukları Forseps ile tutun ve decidua (şekil 1B) ortaya çıkarmak için ayrı çekin.</li>
	<li>Forseps kullanarak, rahim tomurcukları RT PBS steril, içeren yeni bir 10 cm tabak içine aktarın.</li>
	<li>Decidua embriyo çevresinde forseps ipuçları ile düzeltmek.</li>
	<li>Bir kesik decidua anti-mesometrial tarafında iyi forseps (şekil 1 c) ile yapmak ve decidua peel away Forseps ile embriyo (şekil 1 d) ortaya çıkarmak için. Not: Decidua mesometrial pole son derece skarların ve embriyo içerir ve solgun görünür en anti-mesometrial kısmı, onun temel geniş olabilir.</li>
	<li>Embriyo forseps (şekil 1E, F) ile incelemek.</li>
	<li>Bir mikro-kaşık kullanarak, steril PBS bir steril 3.5 cm tabak içinde yer alan bir damla içine embriyo transfer.</li>
	<li>PBS bir mikro-kaşık kullanarak aynı 3.5 cm tabak taze bir damla içine embriyo transfer ve embriyo forseps (şekil 1G, H) kullanarak sarısı sac dan incelemek.</li>
	<li>İsteğe bağlı olarak, (örneğin, Genotipleme Polimeraz zincir reaksiyonu tarafından için) gerekirse, sarısı sac steril tüpe bununla aspirating tarafından transfer ~ 5 µL PBS büyük delik 200 µL pipet ucu içine. -20 ° C'de mağaza Not: sarısı sac hazırlama kontamine anne doku (Yani, parietal sarısı Reichert'ın membran ve ectoplacental koni gibi sac) hatalı Genotipleme sonuçlara yol açabilir. Bu doku kaldırılması embriyo diseksiyon sarısı sac dan önce en kolay yoldur.</li>
</ol>4. allantois diseksiyon ve Eski Utero kültür immobilize α4β1 Integra üzerinde
<ol><li>Allantois iyi forseps (şekil 1 H) kullanarak embriyo posterior sonundaki Bazal ekleme sitesinden kesti.</li>
	<li>İsteğe bağlı olarak, gelişimsel evreleme gerekiyorsa, embriyo somite çift faz kontrast optik ve 5 × ve 10 × amacı ile donatılmış bir mikroskop kullanarak görsel denetim tarafından saymak. Not: Embriyo somite sayma kolaylaştırmak için Forseps ile uzatılabilir. Chorioallantoic eki ve füzyon E 8.0 ve E 9.0 arasında embriyo ≥ 6 somite çiftleri ile oluşur. Dönüm embriyo 6-8 somite çiftleri ile başlar.</li>
	<li>Pipet 40 µL/kuyu % 10 (v/v) fetal sığır serum, L-glutamine ve önceden kaplanmış microtiter plaka wells (bkz. Bölüm 1) gerekli sayıda antibiyotik ile takıma DMEM.</li>
	<li>Bununla aspirating tarafından kayan allantois toplamak ~ 5 µL PBS 200 µL pipet ucu büyük bir delik içine. 1 allantois/iyi transfer.</li>
	<li>Örneğiniçin bir oksijen doku kültürü kuluçka (37 ° C, % 5 CO2) microtiter tabak koyarak allantoides kuluçkaya 6, 12 ve 24 h. Allantois eki ile etkilenmemesi için microtiter plakaları bu zaman puan arasında hareket.</li>
	<li>Her zaman noktada allantois ek (örneğin, hiçbir eki/yüzer allantois kısmi veya tam olarak ekli allantois; tam yayılmış allantois) puan edinildi faz kontrast veya diferansiyel girişim kontrast optik ile donatılmış bir mikroskop kullanarak ve 5 × veya 10 × hedef (Şekil 2). Not: Taze izole ve tam olarak ekli allantoides temsilcisi örnekleri Şekil 2' de gösterilmiştir. Allantoides wildtype C57BL/6J fareler izole distal ucu rahim, verdiği 4-6 h (Bu adlandırılır allantois kısmi ek olarak) içinde ve tüm allantois tam eki içinde elde ~ kültür 12 h. Explant tarafından 18-24 h, basık ve dairesel bir şekil (tam olarak allantois yaymak için denir) ile bir CD31 pozitif vasküler pleksus9,16kabul.</li>
</ol>5. doğrulama Explanted Allantois endotel hücre işaretçisi CD31 için boyama
<ol><li>Tam yayılmış explants kültür orta Aspire edin ve hücreleri % 4 (w/v) paraformaldehyde/PBS 40 µL/kuyu ekleyerek düzeltmek. Not: Paraformaldehyde zehirlidir. Cilt ve göz teması önlemek için koruyucu gözlük ve eldiven giymek.</li>
	<li>RT, 10 min için kuluçkaya sonra süpernatant Aspire edin. PBS 40 µL/kuyu ekleyerek üç kez yıkama ve çamaşır orta yumuşak emici tarafından kaldırın.</li>
	<li>Hücreleri permeabilize ve non-spesifik bağlayıcı siteleri blok 40 µL/iyi % 0.1 (v/v) dik yıkama 30 dk için PBS içinde Triton X-100/0.1% (w/v) BSA/10% (v/v) normal keçi serum ekleyerek iki kez PBS 40 µL/kuyu ekleyerek ve çamaşır orta yumuşak emici tarafından kaldırın. Not: Protokol gecede bu noktada duraklatılmış.</li>
	<li>Hücreleri 20 µL/iyi birincil anti-CD31 antikorların 1:50 % 0,1 (w/v) BSA/PBS içinde seyreltilmiş ekleyerek gecede 4 ° C'de leke PBS 40 µL/kuyu ekleyerek üç kez yıkama ve çamaşır orta yumuşak emici tarafından kaldırın.</li>
	<li>İlişkili birincil antikorlar %0,1 (w/v) BSA/PBS RT. koru microtiter plakaları ışık alüminyum folyo sarma veya yerleştirerek, 1 h için bir karanlık oda içinde ikincil keçi fluorescently etiketli Anti-sıçan antikorlar seyreltilmiş 1: 200 20 µL/kuyu ekleyerek algılamak. PBS 40 µL/kuyu ekleyerek üç kez yıkama ve çamaşır orta yumuşak emici tarafından kaldırın.</li>
	<li>Hücreleri 20 µL/iyi montaj orta ekleyerek gömün. İçin kuluçkaya ~ 1 h montaj orta katılaşmış kadar RT olarak. Microtiter plakaları RT veya ışıktan korunan 4 ° C'de depolayın.</li>
	<li>Görüntü allantois vasküler pleksus 5 × veya 10 × objektif ve uygun floresan filtre ile donatılmış bir floresan mikroskop altında explants. Not: Tam yayılmış allantois CD31 pozitif vasküler pleksus temsilcisi görüntülerini şekil 3' te gösterilmektedir. Diğer işaretler için antikorlar gibi vasküler pleksus Flk-1 karşı Flt, yönetmen kravat-1 veya kravat-2 iyi16olarak kullanılabilir.</li>
</ol>

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Yanlısı yapıştırıcı hücre dışı matriks moleküllerini fibronektin gibi zengin bir kaynağıdır, serum huzurunda allantois explants kolayca çeşitli plastik, cam veya filtre yüzeyler9,16eklemek olacaktır. Böylece, gerçekleştirilen tahlil amacı özellikle bir genetik değişiklik etkisini araştırmak için veya başka bir tedavi allantois eki için immobilize α4β1 ise, non-spesifik bağlayıcı siteleri (örneğin, sığır serum ile ...

Plasenta embriyonik gelişim ve rahim ortamı için vazgeçilmezdir. Endokrin ve bağışıklık organ oksijen, metaboliti ve maternal ve fetal dolaşım ve ayrıca işlevleri arasında besin alışverişini arabirimdeki oluşturmaktadır. Plasental geliştirme bozar endojen veya eksojen hakaret intrauterin büyüme geriliği, gebelik kaybı veya gebelik komplikasyonları, fareler ve insanlar1yol açabilir. Anormal plasental Fizyoloji neden hedeflenen fare mutasyonlar sayısı ile şu anda fare genom Bilişim (MGI) Web sitesinde listelenen 219 genotip2, artmaya devam ederken (http://www.informatics.jax.org/vocab/ mp_ontology / MP:0010038), bu fenotipleri birçoğu iyi mekanik açısından anlaşılmış değil. Genetik değişikliklere ek olarak, küçük molekül uyuşturucu, monoklonal antikorlar, toksinler, patojenler, aşırı metabolitleri veya çeşitli çevresel aracıları plasenta geliştirme ve işlev3,4,5 etkileyebilir , 6 , 7. henüz, plasenta geliştirme modeli eleştirel adımları azdır basit vitro deneyleri.
Allantois (göbek kordonu gelişimsel habercisi) embriyo posterior ucundan doğru koryon8büyür bir tomurcuk olarak ortaya çıkıyor zaman fare plasental geliştirme erken midgestation içinde başlatılır. Allantoic tomurcuk dış tabakası hücrelerde Chorioadhesive eki ve allantois koryon (chorioallantoic "füzyon") yüzeyi ile yayılan aracılık. Koryon villus içine Labirent, besin ve oksijen anne kan yakından bitişik sinusoids ve embriyonik damarları9 arasında değişen nerede iç plasental katman oluşturmak üzere allantois damarlara büyür içine daha sonra pas geçiyor ,10.
Allantois eki koryon için labirent oluşumu içinde belgili tanımlık ilk ve önemli adım, ve kusurları bu süreçte embriyonik ölümcül en sık karşılaşılan nedenleri arasında midgestation1'. Her ne kadar bir dizi fare mutantlar var nerede chorioallantoic eki10gerçekleşmesi başarısız olur ve MGI veritabanı şu anda anormal chorioallantoic füzyon (http:// tarafından karakterize 108 fare mutantlar listeler tarif edilmiştir www.informatics.Jax.org/Vocab/mp_ontology/MP:0002824), vasküler hücre adezyon molekülü-1 (VCAM-1, allantoic mesoderm ifade) ve (olan koryonik mesothelium ifade α4β1 integrin arasındaki hücreler arası etkileşimi extraembryonic mesoderm türetilmiş) chorioallantoic eki ve füzyon11,12,13için vazgeçilmez gibi görünüyor. İmmunohistokimyasal bütün Dağı yaban tipi embriyoların boyama olduğunu göstermiştir VCAM-1 yaklaşık embriyonik gününde (E) 7.5 distal üçte içinde allantoic SAP ifade edilir13 (1-4 somite sahne) ve onun ifade yüksek kalır chorioallantoic eki ve füzyon11,12sırasında-. Koryon allantoic eki başarısızlık genellikle rahim tomurcukları histolojik analizler tarafından algılanır. Ancak, allantois boyutları fizyolojik arasında ve aynı gelişimsel sahne14çöp içinde bile çok değişkendir ve fiziksel temas yapmak için yeterli bir boyuta ulaştığında allantois sadece için koryon ekleyebilirsiniz. Bu doğal değişkenlik yansıtan, chorioallantoic ek bir ara arasında gerçekleşir ~ E 8.0 ve E 9.0 rahim içindeve bu işlem Histoloji tarafından istatistiksel olarak güvenilir bir şekilde değerlendirilmesi bağlıdır bu nedenle çok sayıda analizi conceptuses uygun gelişim aşamasında yeterli örnekler almak için.
Burada, biz daha az allantois boyutuna bağlıdır allantoic eki rahim değerlendirmek için bir yöntem açıklanmaktadır. Biz hamile fareler uteri fare embriyo ve onların allantoides diseksiyon göstermek ~ 8 gün coitum post (dpc; dpc 0,5 belirtir bir vajinal fiş tespiti), ve allantois sonraki kültür üzerinde immobilize α4β1 explants integrinler. Bu yöntem ekler ve birden çok allantois explants paralel ve farklı her başarılı zaman noktalarda davranışını yayılan bir hızlı, işlevsel değerlendirme sağlar. Protokol kullanılabilir için ekran allantois eki ex vivohedeflenen mutasyonlar, uyuşturucu veya çeşitli çevresel faktörlerin etkileri.

<table border="0" cellpadding="0" cellspacing="0" width="1607">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">C57BL/6J wildtype mice</td>
			<td style="width:256px;">The Jackson Laboratory</td>
			<td style="width:119px;">Stock No:&#160;000664</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 % (v/v) Ethanol</td>
			<td style="width:256px;">VWR International</td>
			<td>930031006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>11000-12</td>
			<td style="width:915px;">Forceps used to open the abdominal cavity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Micro dissecting forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td>Dumont # 5 medical biology forcepts with fine, sharp tip.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fine scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>14094-11</td>
			<td>Straight blades.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>15012-12</td>
			<td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microspoon</td>
			<td style="width:256px;">Carl Roth</td>
			<td>AT18.1</td>
			<td>5 mm Spoon diameter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microtiter plates</td>
			<td style="width:256px;">ibidi</td>
			<td style="width:119px;">81501</td>
			<td>We use uncoated, sterile angiogenesis slides (internal volume 50 &#181;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-a4b1-mediated binding to plastic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">10 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">3.5 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150288</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large orifice pipette tips&#160;</td>
			<td style="width:256px;">Biozym</td>
			<td style="width:119px;">VT0140X</td>
			<td>Low binding pipette tips, 200 &#181;L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a4b1 integrin</td>
			<td style="width:256px;">R&#38;D Systems</td>
			<td style="width:119px;">6054-A4</td>
			<td>Murine a4b1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>Without Ca2+ and Mg2+.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:319px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-03600</td>
			<td style="width:915px;">The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO3 but no L-glutamine, which is added separately.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:319px;">Penicillin/Streptomycin</td>
			<td style="width:256px;">Gibco (ThermoFisher Scientific)</td>
			<td style="width:119px;">15140-122</td>
			<td>100 x (10,000 U/mL Penicillin and 10,000 &#181;g/mL streptomycin) stock solution.&#160;Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">L-Glutamine</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-80100</td>
			<td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fetal bovine serum&#160;</td>
			<td style="width:256px;">Biochrom</td>
			<td style="width:119px;">S 0115</td>
			<td>We use heat-inactivated FBS (heated to 56 &#176;C for 30 min with mixing to inactivate complement).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin (BSA)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">A7030</td>
			<td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &#181;m pore size and low protein-binding membrane, and store aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">para-Formaldehyde</td>
			<td style="width:256px;">Carl Roth</td>
			<td>0335.2</td>
			<td style="width:915px;">To make a formaldehyde solution in PBS, weigh para-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &#176;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &#176;C for approximately four weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Triton X-100</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">X100</td>
			<td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Normal goat serum</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>G9023</td>
			<td>To block non-specific binding of antibodies in immunofluorescence analyses.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a-CD31 Antibodies</td>
			<td style="width:256px;">BD Biosciences</td>
			<td>550274</td>
			<td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary goat anti-rat antibodies</td>
			<td style="width:256px;">Thermo Fisher</td>
			<td>A-11006</td>
			<td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Mowiol 4-88</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>81381</td>
			<td>Mounting medium for cytochemistry. MW ~31,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Labovert&#160;</td>
			<td style="width:256px;">Leitz</td>
			<td style="width:119px;">Labovert&#160;</td>
			<td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">M80</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">M80</td>
			<td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">DM4000B</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">DM4000B</td>
			<td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Digital camera</td>
			<td style="width:256px;">JVC</td>
			<td style="width:119px;">KY-F75U</td>
			<td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:319px;">Confocal microscope</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">SP5</td>
			<td style="width:915px;">Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td>
		</tr>
	</tbody>
</table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

Plasenta büyüme ve memeli embriyo gelişimi için önemlidir. Bu nedenle, çok sayıda genetik değişiklikler ve plasenta geliştirme veya işlev rahatsız büyük olasılıkla aynı zamanda çevre hakaret erken gebelik kaybı fareler ve insanlar neden olabilir. Yine de, basit vitro deneyleri plasenta oluşumu üzerindeki olası etkileri için ekran için eksik vardır. Burada, allantois ekin için koryon oluşur plasenta oluşumu ilk ve önemli adımda modelleme ele. Biz hızla allantoic explants chorio mimetic substrat hizmet vermektedir immobilize α4β1 Integra üzerinde eki değerlendirmek için bir yöntem açıklanmaktadır. Bu vitro yaklaşım eki ve birden çok allantois explants farklı ardışık zaman noktalarda davranışını yayılan nitel bir değerlendirme sağlar. Protokol hedeflenen fare mutasyonlar, uyuşturucu veya gebelik komplikasyonları veya fetal kayıp allantois eki ex vivoolarak bağlı çeşitli çevresel faktörlerin etkisini araştırmak için kullanılır.

Bu iletişim kuralı yalıtmak ve fare allantoides ex vivoexplant yöntemi açıklanır ve in vivo chorioallantoic füzyon sırasında α4β1 integrin allantois eki, kritik bir süreç değerlendirme nitel ayrıntıları. Şekil 1A-H temsilcisi sonuçlar allantois yalıtım hamile rahim başlayarak birbirini izleyen adımları göstermektedir. Rahim boynuzları, yağ dokusu ve gemiler (?...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Biz bir vitro tahlil modeli chorioallantoic ekte, plasenta oluşumu ilk adımda açıklanmaktadır. Protokol immobilize α4β1 Integra üzerinde fare allantoides diseksiyon ve explant kültür gösterir. Allantois ek olarak önceden belirlenmiş zaman noktalarda değerlendirilir.

Diseksiyon ve Explant kültür Allantoic eki Vitro analizi için fare Allantois ve

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

dissection-explant-culture-murine-allantois-for-vitro-analysis

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Medicine

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

7.8K Görüntüleme.  University of Würzburg. Biz bir vitro tahlil modeli chorioallantoic ekte, plasenta oluşumu ilk adımda açıklanmaktadır. Protokol immobilize α4β1 Integra üzerinde fare allantoides diseksiyon ve explant kültür gösterir. Allantois ek olarak önceden belirlenmiş zaman noktalarda değerlendirilir.

Video: Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

Proteomik Analizi İnsan Vitreus Gövde Elemanları diseksiyonu

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Tıp

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

İlköğretim İnsan Fibroblastlar türetilmesi için Cilt Punch Biyopsi Eksplantın Kültür

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Farelerde Taksanlar Farmakokinetik ve Farmakodinamik Analiz Karotis Arter İnfüzyonlar

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

Vitreodynamic Analiz Tüm Vitreus Mizah Diseksiyon

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

Mürin Prostat Mikro-diseksiyon ve Cerrahi Kastrasyon

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Optimize Analizi<em&gt; İn Vivo</em&gt; ve<em&gt; İn Vitro</em&gt; Karaciğer Steatoz

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Fare pankreas diseksiyon histolojik analiz ve metabolik profil oluşturma

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Yenidoğan Murin Koklear Explant Tekniği<em&gt; In vitro</em&gt; İşitme Araştırmalarında Tarama Aracı

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortopathy aort anevrizması β-aminopropionitrile-indüklenen ve diseksiyon, fare modeli nicel mikro-CT Analizi

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...