K. Gill, fare yetiştirme ve bakımı ile ilgili yardım ve laboratuvar yönetimi için teşekkür ederiz. Teknik yardım için Mukoyama laboratuar üyeleri de teşekkür ederiz. Finansman, Sağlık Naitonal Enstitüleri İntramural Araştırma Programı tarafından sağlanmıştır.

1. Toplama Fare Embriyonik Ekstremite Skin (E13.5 ~ E17.5) <ol><li> Euthanize Onaylanan prosedür kadın takılı. Onaylı hayvan protokole göre, kadınlarda CO 2 maruz tarafından ötenazi ve sonra servikal dislokasyon ile güvence altına. Emici kağıt havlu üzerine yatırın ve hayvan sıkmak şişeden% 70 EtOH / H 2 O iyice ıslatın. </li><li> Rahim sağlam inceleyin ve kan yıkamak için buz Hanks &#39;Dengeli Tuz Çözeltisi (HBSS) içeren 100 x 15 mm Petri kabı yerleştirin. </li><li> Embriyo ayrı ve teşrih. Embriyo çok ince amniyon çıkarın. </li><li> Her embriyo genotip gerekiyorsa (Seçenek) 35 x 10 mm Petri kabında tek bir embriyo parçalara ayır. Disseke kuyruk genotipleme için 0,2 ml PCR tüpü aktarılır. </li><li> Embriyonun ön ayakları kesiliyor ve transferi 24 plaka 2 ml Phosphate Buffer Saline (PBS) buz gibi taze% 4 Paraformaldehit (PFA) içeren bir halka forseps ön ayakları. </li><li> 4 ° C gecede Nutator Mikser nazik karıştırma ön ayakları sabitleyin. </li><li> Ertesi gün, PFA çıkartın ve oda sıcaklığında Nutator Mikser nazik karıştırma ile 2 ml PBS 5 dakika boyunca üç kez yıkayın. </li><li> % 100 metanol (MeOH) ön ayakları transferi ve -20 ° C&#39;de enzim dondurucu (kritik sıcaklık kontrolü ve otomatik buz çözme fonksiyonu olmadan dondurucu) saklayabilirsiniz. İlköğretim antikorlar% 100 MeOH tedaviden sonra Tablo 1 iş listelenmiştir. </li><li> Ince cımbız kullanarak ön ayakları kapalı cilt Peel. Ekstremite cilt, kurutulmuş, uzuv kolayca ayrılmalıdır. İlk uzuv yukarı bakacak ventral tarafı (avuç içi tarafında) yerleştirin. Sonra videoda gösterildiği gibi ince cımbız kullanarak, deri kesme. Sonraki herhangi bir zarar vermeden cildi nazikçe sıyrılarak, tüm bacak çevresinde teşrih. </li></ol> 2. Tüm montaj Ekstremite Skins İmmünohistokimyasal Boyama <ol><li> MeOH / PBT (PBS +% 0.2 Triton X-100) (% 75,% 50,% 25) için her biri 5 dakika aşamalı bir dizi kuluçka 5ml polipropilen yuvarlak alt tüp bacak derileri rehidrate. Çözümler alışverişi ekstremite derileri zarar vermemek için dikkatli olunması gerektiğini unutmayın. </li><li> , Oda sıcaklığında Nutator Mikser nazik karıştırma PBT 5 dakika için iki kez yıkayın. </li><li> Nutator Mikser nazik karıştırma ile 2 saat eşek sekonder antikor için% 10 keçi sekonder antikorlar serum / PBS +0.2% TX100 engelleme tampon Keçi veya% 10 Eşek serum / PBS +0.2% TX100 engelleme tampon ya bacak derileri Blok oda sıcaklığında bekletin. </li><li> Engelleme tampon birincil antikor 800μl (Tablolarda listelenen uygun dilüsyon) (2ml mikrosantrifüj tüp içine bir halka forseps ile 35 x 10 mm Petri kabı ve transfer bacak deriler% 10 ya da koyun keçi serum / PBS 0,2 % TX100 veya% 10 Eşek serum / PBS +0.2% TX100). 4 ° C gecede Nutator Mikser nazik karıştırma ile bacak derileri inkübe edin. Farklı türleri (örneğin, sıçan monoklonal antikor + tavşan poliklonal antikor) türetilen multipl primer antikorlar aynı anda kullanılabilir olabileceğini unutmayın. </li><li> Ertesi gün, yıkama tampon 4ml (ya% 2 Keçi serum / PBS +0.2% TX100 veya 2, yuvarlak alt 5ml polipropilen tüp içine bir halka forseps ile 35 x 10 mm Petri kabı ve transfer ekstremite derileri yerde % Eşek serum / PBS +0.2% TX100). </li><li> Nazik oda sıcaklığında Nutator Mikser karıştırma ile 15 dakika süreyle beş kez yıkayın. </li><li> 800μl engelleme tampon sekonder antikor (ya% 10 Keçi serum / PBS +0.2% TX100 veya% 10 Eşek serum / 2ml-mikrosantrifüj tüp içine bir halka forseps ile 35 x 10 mm Petri kabı ve transfer bacak derileri koyun PBS +0.2% TX100). Genelde biz Jackson ImmunoResearch (Cy3, Cy5, 1:300 dilüsyonları) veya Invitrogen (Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 633, 1:250 dilüsyon) ikincil antikorlar kullanır. 0.22μm PVDF membran şırınga filtreleri kullanarak sekonder antikor toplu parçacıkları çıkarmak için ikincil antikor çözüm Filtre. Oda sıcaklığında Nutator Mikser nazik karıştırma ile 1 saat karanlık veya alüminyum folyo ile sarılmış bacak derileri inkübe edin. Farklı türlerden elde edilen farklı floresan konjuge sekonder antikorlar aynı anda kullanılabilir olabileceğini unutmayın. </li><li> Bacak derileri koyun yıkama tampon 4ml (% 2 Keçi serum / PBS 0,2% ya TX100 veya% 2 Eşek serum / PBS ile 5ml polipropilen yuvarlak alt tüpe bir halka forseps ile 35 x 10 mm Petri kabı ve transfer 0,2% TX100). Koyu veya yumuşak oda sıcaklığında Nutator Mikser karıştırma alüminyum folyo ile sarılmış 15 dakika süreyle beş kez yıkayın. </li><li> (Çekirdeği karşı counterstaining için Seçenek) yıkama tampon 4ml (ya% 2 Keçi serum / PBS 0,2% TX100 veya% 2 Eşek serum / PBS +0.2% TX100) için-Pro-3 (Invitrogen T3605 ile bacak derileri inkübe , koyu veya oda sıcaklıklarda Nutator Mikser nazik karıştırma ile 10 dakika süreyle alüminyum folyo ile sarılır 1:3000 dilüsyon)yeniden. Daha sonra, nazik karıştırma ile alüminyum folyo ile karanlık veya sarılmış yıkama tampon 4ml (% 2 Keçi serum / PBS TX100 veya% 2 Eşek serum / PBS +0.2% +0.2% TX100 ya) ile 5 dakika boyunca üç defa yıkamak oda sıcaklığında Nutator Mikser. Çekirdekleri için güçlü ve spesifik boyanması için pro-3 için belirli bir emisyon (HeNe 633 nm eksitasyon) görülmektedir unutmayın. </li></ol> 3. Slayt Ekstremite Skins Montaj <ol><li> 35 x 10 mm Petri kabında bacak derileri yerleştirin. Beyazlatma kapsamlı fotoğraf çekmemek için düşük aydınlatma stereomicroscope altında ince bir cımbız kullanarak derileri iç tabakası toz, kristaller, elyaf çıkarın. </li><li> Bir halka forseps yapışkan mikroskobik slayt bacak derileri aktarın. Slayt (yani lamel doğru) yukarı doğru uzanan iç tabakası ile derileri yerleştirin. Derileri titizlikle cımbız kullanarak Düzleştir&#39;i kaldırmak ve Kimwipe tarafından tampon yıkama-over taşımak. </li><li> Hava kabarcıkları olmadan, anti-solmaya montaj medya monte edin. Biz 25 &quot;x25&quot; lamel kullanın. Karanlıkta düz bir yüzey üzerinde Cure (örneğin, örnekleri görüntülemeden önce oda sıcaklığında karanlık bir gece olarak yerleştirilir Altın reaktif uzatmak kullanılarak monte). Uzun süreli depolama için, 4 slayt lamel ve mağaza mühür ° C </li></ol> 4. Konfokal Mikroskop <ol><li> Fluorophores için uygun lazerler kadar ayarlayın. Biz, Argon 488nm (Alexa Fluor 488 ve GFP), DPSS 561nm (Alexa Fluor 568 ve Cy3) ve HeNe 633nm (Alexa Fluor 633, Cy5 ve To-Pro-3 için) olmak üzere üç lazer kaynakları ile Leica TCS SP5 konfokal mikroskop . </li><li> Tüm boyalar, çift ya da üçlü lekeli örneklerinde aynı zamanda heyecanlı olacağı crosstalk önlemek veya azaltmak için sıralı tarama aracını kullanın. Sıralı tarama modu, sıralı olarak kaydedilir. </li><li> Uyarma için floresan boyalar ve lazer hakkında daha fazla genel bilgi Hibbs (2004) tarafından &quot;Biyologlar için Konfokal Mikroskop&quot; kurulmuş olabilir </li></ol> 5. Temsilcisi Sonuçlar Pan-endotelyal hücre işaretleyici karşı antikorlar E15.5 fare ön ayakları deride Tüm montaj üç-etiket konfokal immnofluorescence mikroskopi PECAM-1 (Şekil 1A-C, D, mavi), nörofilaman işaretleyici 2H3 (Şekil 1A yeşil) ve ekstremite cilt 2H3 + periferik sinirleri ile uyumlu αSMA + arterlerin bir karakteristik dallanma deseni, düz kas hücre belirteci αSMA (Şekil 1A, B kırmızı) ortaya çıkardı. Dallanma kan damarı yanı sıra, bu uzuv deri vaskülatürü modeli lenfatik endotel hücre işaretleyici LYVE-1 (Şekil 1D, E kırmızı) ve Neuropilin2 (NRP2) (Şekil 1D, F yeşil antikorlar kullanılarak lenfatik damar dallanma desenlendirme incelemek için kullanılır. .) <img src="/files/ftp_upload/2620/2620fig1.jpg" alt="Şekil 1" /> Şekil 1 (AC) Arterler embriyonik ekstremite cilt periferik sinir ile aynı hizaya getirin. Pan-endotelyal hücre belirteci PECAM-1 (AC, mavi), nörofilaman işaretleyici 2H3 (A, yeşil) ve düz kas hücre işaretleyici αSMA (A ve B, kırmızı karşı antikorlar ile tüm montaj üçlü-etiket konfokal immnofluorescence mikroskopi ) gösterilir. E15.5, 2H3 + periferik sinirlerin (açık ok) αSMA tarafından karşılanmaktadır arterler (ok başları) + düz kas hücreleri ile ilişkilendirir. (DF) embriyonik ekstremite cilt Lenfatik damarsal. Pan-endotelyal hücre belirteci antikorlar PECAM-1 (D, mavi), lenfatik endotel hücre işaretleyici LYVE-1 (D ve E, kırmızı) ve Neuropilin2 (NRP2) (D ve F, yeşil Triple-etiket konfokal immnofluorescence mikroskopi ) gösterilir. LYVE-1 de makrofajlar (açık ok başları) bir alt kümesi ile ifade edilir ise, lenf damarları, LYVE-1 ve NRP2 her iki tarafından görüntülenmiştir. Ölçek çubuğu: 100μm.

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+distinction+and+angiogenic+interaction+between+embryonic+arteries+and+veins+revealed+by+ephrin-B2+and+its+receptor+Eph-B4.">Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.</a> Cell. 93, 741-753 (1998).</li><li>Gerety, S. S., Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+mutant+phenotypes+of+the+receptor+EphB4+and+its+specific+transmembrane+ligand+ephrin-B2+in+cardiovascular+development.">Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.</a> Mol Cell. 4, 403-414 (1999).</li></ol>

Organlara yeterli oksijen ve besin kaynakları nedeniyle vasküler sistem, embriyogenez sırasında yanı sıra yetişkinler organ bakım ve üreme fonksiyonları için organ gelişimi için çok önemlidir. Uygun damar ağı, önceden mevcut olan kapiller ağ çok dallı ve hiyerarşik yapıları ile yeniden düzenlenmiş olan anjiyogenez karmaşık ve çok adımlı işlemler ile kurulmuştur. Birçok eseri çeşitli moleküller bu süreçlere dahil olduğu gösterilmiş olsa da, organ veya bileşenleri vasküler dalla...

Antikorlar Pan-endotelyal hücre belirteci <table border="1"><tbody><tr><td> Antikor </td><td> Tür </td><td> Şirket </td><td> Katalog # </td><td> Çalışma şartları </td></tr><tr><td> PECAM-1 </td><td> Ermeni, hamster (M) </td><td> Chemicon </td><td> MAB1398Z </td><td> 1:100 dilüsyon # 1 </td></tr><tr><td> PECAM-1 </td><td> Rat (M) </td><td> BD </td><td> 553369 </td><td> 1:300 dilüsyon </td></tr><tr><td> VEGFR2 </td><td> Rat (M) </td><td> eBioscience </td><td> 14-5821-82 </td><td> 1:200 oranında seyreltildiğinde </td></tr><tr><td> CD34 </td><td> Rat (M) </td><td> eBioscience </td><td> 13-0341 </td><td> 1:300 dilüsyon </td></tr><tr><td> Kollajen IV. </td><td> Tavşan (P) </td><td> Abd Serotec </td><td> 2150-1470 </td><td> 1:300 dilüsyon # 2 </td></tr></tbody></table> Arter endotel hücre işaretleyici <table border="1"><tbody><tr><td> Antikor </td><td> Tür </td><td> Şirket </td><td> Katalog # </td><td> Çalışma şartları </td></tr><tr><td> Neuropilin1 </td><td> Tavşan (P) </td><td> Alex Kolodkin laboratuvar # 3 </td><td></td><td> 1:3000 dilüsyon </td></tr><tr><td> Unc5H2 </td><td> Keçi (P) </td><td> Ar-Ge </td><td> AF1006 </td><td> 1:200 oranında seyreltildiğinde </td></tr></tbody></table> Venöz endotel hücre işaretleyici <table border="1"><tbody><tr><td> Antikor </td><td> Tür </td><td> Şirket </td><td> Katalog # </td><td> Çalışma şartları </td></tr><tr><td> EphB4 </td><td> Keçi (P) </td><td> Ar-Ge </td><td> AF446 </td><td> 1:100 dilüsyon </td></tr></tbody></table> Lenfatik endotel hücre işaretleyici <table border="1"><tbody><tr><td> Antikor </td><td> Tür </td><td> Şirket </td><td> Katalog # </td><td> Çalışma şartları </td></tr><tr><td> LYVE-1 # 4 </td><td> Tavşan (P) </td><td> Abcam </td><td> ab14917 </td><td> 1:200 oranında seyreltildiğinde </td></tr><tr><td> LYVE-1 # 4 </td><td> Rat (M) </td><td> MBL </td><td> D225-3 </td><td> 1:200 oranında seyreltildiğinde </td></tr><tr><td> Prox-1 </td><td> Tavşan (P) </td><td> Chemicon </td><td> AB5475 </td><td> 1:1000 dilüsyon </td></tr><tr><td> Prox-1 </td><td> Keçi (P) </td><td> Ar-Ge </td><td> AF2727 </td><td> 01:50 seyreltme </td></tr><tr><td> Neuropilin2 </td><td> Tavşan (P) </td><td> Hücre Sinyalizasyonu </td><td> 3366 </td><td> 1; 100 seyreltme </td></tr><tr><td> Podoplanin </td><td> Suriye hamsteri (M) </td><td> Hibridoma Bankası </td><td> 8.1.1 </td><td> 1:200 oranında seyreltildiğinde </td></tr></tbody></table> Düz kas hücre / pericyte işaretleyici <table border="1"><tbody><tr><td> Antikor </td><td> Tür </td><td> Şirket </td><td> Katalog # </td><td> Çalışma şartları </td></tr><tr><td> αSMA-Cy3 </td><td> Fare (M) # 5 </td><td> Sigma </td><td> C-6198 </td><td> 1:500 dilüsyon # 6 </td></tr><tr><td> NG2 </td><td> Tavşan (P) </td><td> Chemicon </td><td> AB5320 </td><td> 1:200 oranında seyreltildiğinde </td></tr><tr><td> SM22α </td><td> Tavşan (P) </td><td> Abcam </td><td> ab14106 </td><td> 1:200 oranında seyreltildiğinde </td></tr></tbody></table> GFP muhabiri için Antikorlar <table border="1"><tbody><tr><td> Antikor </td><td> Tür </td><td> Şirket </td><td> Katalog # </td><td> Çalışma şartları </td></tr><tr><td> GFP </td><td> Tavşan (P) </td><td> Invitrogen </td><td> A11122 </td><td> 1:300 dilüsyon </td></tr><tr><td> GFP </td><td> Rat (M) </td><td> Nacalai tesque </td><td> 04404-84 </td><td> 1:1000 dilüsyon </td></tr><tr><td> GFP </td><td> Civciv (P) </td><td> Chemicon </td><td> P42212 </td><td> 1:300 dilüsyon </td></tr></tbody></table> LacZ muhabiri için Antikorlar <table border="1"><tbody><tr><td> Antikor </td><td> Tür </td><td> Şirket </td><td> Katalog # </td><td> Çalışma şartları </td></tr><tr><td> ß-gal </td><td> Tavşan (P) </td><td> MP Biyomedikal </td><td> 55976 </td><td> 1:5000 dilüsyon </td></tr><tr><td> ß-gal </td><td> Keçi (P) </td><td> Abd Serotec </td><td> 4600-1409 </td><td> 1:500 dilüsyon </td></tr><tr><td> ß-gal </td><td> Civciv (P) </td><td> Abcam </td><td> ab9361 </td><td> 1:200 oranında seyreltildiğinde </td></tr></tbody></table> Periferik akson için Antikorlar <table border="1"><tbody><tr><td> Antikor </td><td> Tür </td><td> Şirket </td><td> Katalog # </td><td> Çalışma şartları </td></tr><tr><td> 2H3 </td><td> Fare (M) # 7 </td><td> Hibridoma Bankası </td><td> 2H3 </td><td> 1:200 oranında seyreltildiğinde </td></tr><tr><td> Tuj1 </td><td> Fare (M) # 8 </td><td> Covance </td><td> MMS-435P </td><td> 1:500 dilüsyon </td></tr><tr><td> Peripherin </td><td> Tavşan (P) </td><td> Chemicon </td><td> AB1530 </td><td> 1:1000 dilüsyon </td></tr></tbody></table> Schwann hücreleri göç için antikorlar <table border="1"><tbody><tr><td> BFABP </td><td> Tavşan (P) </td><td> Thomas Müller laboratuvar # 9 </td><td></td><td> 1:3000 dilüsyon </td></tr></tbody></table> (P): poliklonal antikor (M): monoklonal antikor # 1: keçi anti-Ermeni hamster Cy3 (Jackson ImmunoResearch 127-165-160) antikor ikincil bir antikor olarak kullanılmalıdır. # 2: Kollajen IV antikor in situ hibridizasyon sonra kan damarlarını tespit etmek için kullanılabilir. # 3: Neuropilin1 antikor nazik Alex Kolodkin Johns Hopkins Üniversitesi laboratuvarı tarafından sağlanmaktadır. Koyun anti-insan Neuropilin1 antikor, henüz test edilmemiş olmasına rağmen Ar-Ge (AF3870) kullanılabilir. # 4: LYVE-1 antikorları da embriyonik deri makrofaj bir alt kümesi algılar. # 5: anti-αSMA antikor fare IgG2a monoklonal antikor. # 6: Cy3 konjuge αSMA antikor sn birlikte oda sıcaklığında 1 saat inkübediğer primer antikorları için ikincil antikorlar. # 7: 2H3 antikor nörofilaman karşı fare IgG1 monoklonal antikor. # 8: Tuj1 antikor Nöron-spesifik sınıf III beta-tubulin karşı fare monoklonal antikor IgG2a. # 9: BFABP (beyin özel yağ asidi bağlayıcı protein) antikoru nazik Thomas Müller Max-Delbrück Merkezi Moleküler Tıp laboratuarı tarafından sağlanmaktadır.

whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo

Tüm montaj immünohistokimyasal analiz görüntüleme için tüm damarsal morfolojilerinden dallanma hücresel mekanizmaları anlamak için çok önemlidir. Biz önceden varolan ilkel kapiller pleksus hiyerarşik dallı bir damar ağına yeniden organize olduğu damar gelişimini incelemek için ekstremite deri vaskülatürü modeli geliştirdik. Birden fazla etiketleme ile tüm montaj konfokal mikroskopi belirli floresan belirteçleri kullanarak, endotel hücreleri perisitlerden ve düz kas hücreleri de dahil olmak üzere, sağlam kan damarlarının yanı sıra hücresel bileşenleri, güçlü görüntüleme için izin verir. Genetik çalışmalar ile bu uzuv deri vaskülatürü modeli gelişmeler, vasküler gelişimi ve desenlendirme moleküler mekanizmaları anlamak düzeldi. Ekstremite deri vaskülatürü modeli, periferik sinirleri arterlerin farklılaşma ve desenlendirme için mekansal bir şablon nasıl çalışmak için kullanılır olmuştur. Bu video makalede, vasküler gelişim sürecini takip edebilecek vaskülarize embriyonik organları için geçerli olan vasküler spesifik antikorlar ve floresan ikincil antikorlar, sağlam kan damarları leke için basit ve sağlam bir protokol tanımlamaktadır.

Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Biz bir bütün montaj immünohistokimya tanıtmak ve lazer fare embriyonik ekstremite cilt karmaşık damar ağı oluşumunu analiz etmek için birden fazla etiketleme ile konfokal mikroskopi tarama.

Embriyonik Ekstremite Cilt damarları Tüm montaj İmmünohistokimyasal Analizi: Embriyo vasküler dallanma Morphogenesis Eğitim Model Sistemi

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

whole-mount-immunohistochemical-analysis-for-embryonic-limb-skin

Research

JoVE Journal

Biology

17.5K Görüntüleme.  National Heart, Lung, and Blood Institute, National Institutes of Health. Biz bir bütün montaj immünohistokimya tanıtmak ve lazer fare embriyonik ekstremite cilt karmaşık damar ağı oluşumunu analiz etmek için birden fazla etiketleme ile konfokal mikroskopi tarama.

Video: Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Erken Civciv Embriyolar in situ Hibridizasyon Çift Tüm Dağı

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

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Chick Tüm Dağı Antikor Boyama Yöntemi

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebra balığı Tüm Dağı Yüksek Çözünürlüklü Çift Floresan Yerinde Hibridizasyon

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Fare Embriyonik Akciğer Kültür, Dallanma Moleküler Mekanizmalar değerlendirin Bir Sistem

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

KRİYOPREZERVE Numune Kurtarma için Do-It-Yourself Aygıt yanlışlıkla Kriyojenik Depolama Tankları içine Dropped

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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 ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; Yerinde</emTüm Dağı Embriyolar ve Hücre histolojisi&gt; Hibridizasyon Deniz Elasmobranchs İçin Uyarlanmış

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Aktif Kaspaz 3 Algılama Tüm montaj immünofloresans tarafından Zebra balığı Embriyolar Apaptozun Analizi

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...