Biz Beninio Jomok Zebra balığı yetiştiriciliği yaptığı yardım için teşekkür ederim. Bu çalışma Ulusal Sağlık Enstitüsü, HL81753 tarafından finanse edilmektedir.

1. Slayt hazırlanması ve nemlendirilmiş odasına <ol><li> Nemlendirilmiş odası boşaltılmış bir ucu kutusu gibi bir kutu yapılabilir. Odası ve örnekleri ışıktan korumak için alüminyum folyo ile kapağı sarın. </li><li> Odanın içinde, kalbinde örnekleri kurumasını önlemek odanın içinde nem, korumak için bir yığın ıslak kağıt havlu koyun. Slaytlar için bir raf olarak kağıt havlu üstüne küçük bir mikroplak ayarlayın. </li><li> Kalp 5x5 mm boyutunda numuneler için kuyu immEdge kalem kullanarak bir mikroskop lamı yüzeyinde ya çizgiler veya daire çizin. Slaytları kurumasını bekleyin. </li><li> Nemlendirilmiş odasında slaytlar koyun ve her iyi 50ul taze% 4 formaldehit ekleyin. </li></ol> 2. Embriyonik kalp diseksiyonu <ol><li> Balık balık durdurmak vücut hareketleri kadar yaklaşık 1 dakika boyunca% 0.4 tricaine içeren E3 yumurta su 7 embriyo anestezisi ama yine de normal kalpdayak. </li><li> Diseksiyon mikroskobu sahnede içbükey bir mikroskop lamı koyun. Concaved iyi embriyolar transfer. </li><li> Kalp açıkça ortaya çıkarmak için diseksiyon mikroskop ışık kombinasyonu ayarlayın. Kişisel tercihinize bağlı olarak, bu karanlık alan veya DIC gibi polarize ışık durumu olabilir. </li><li> Sonra kuru, iki insülin şırıngaları% 75 etanol ile temizleyin. </li><li> Diseksiyon mikroskobu, kalp yüksek büyütme ve odak ayarlayın. Fiziksel kafasına yakın yumurta sarısı-hücre gruplarının sınır içine bir iğne ucu takarak bir embriyo düzeltin. Başka bir iğne yakın kalp bölgeye yerleştirin ve hızlı bir şekilde kalp çekin. Kalp embriyo tamamen ayrılmış değildir, kalp ve yumurta sarısı arasında kalan bağlı doku kesti. </li><li> Mikroskop ayrılan kalp düşük büyütme ve odak ayarlayın. 10μl pipetteman kullanın ve 1μl hacim olarak ayarlayın. Çizin veya dokunmatik pipetteman ucu uygulaizole kalp içinde veya ucu yüzeye bağlı ya olacak, böylece kalp örnek. </li><li> % 4 formaldehit hazırlanan slaytlar pipet ucu batırın ve çözüm kalp serbest pipetle piston bastırın. Su yüzey gerginliği de pipet çözüm kalp serbest bırakmak için yardımcı olur. Her kuyuda 3 kalpleri daha az koyun. </li></ol> 3. Immün <ol><li> Odasına kapatın ve yumuşak bir sallanan hareketi ile bir çalkalayıcı üzerine koydu. Oda sıcaklığında 20 dakika boyunca kalp örnek sabitleyin. </li><li> Slaytların arka su damlacıklarını ve tampon alışverişi için bir diseksiyon mikroskobu aşamasında slayt koymak. </li><li> Her bir kuyunun boş bir bölgeye, bir 10μl pipetteman ucu batırın ve kalp örneklerinden ucu mümkün olduğunca tutmak. Çözüm ucu içine çekilir kalpleri su akışı ile hareket edebilir, ucuna takılı kalp önlemek için tedbirleri almak. DeğiştirmekGerekirse ucu yerelleştirme. </li><li> Çözüm bir doku üzerine atın ve kalp örnek hava kabarcıklarının varlığı kolayca kayıp olduğundan uç kabarcıkları üreten kaçının. Kalp örnekleri kısa bir süre için kısmen kuru tutulması, kalpleri slaytlar bağlı kalmanıza yardımcı olabilir. </li><li> Kalp örnek kapak ve 5 dakika boyunca yavaş bir sallanan hareketi nemlendirilmiş odasında inkübe 50μl PBST ekleyin. Durulama bir kez daha tekrarlayın. Bu adımdan sonra, kalp örnekleri nemlendirilmiş, 4 oda 1 haftaya kadar ° C saklanabilir </li><li> PBST oda sıcaklığında 1 saat süreyle% 10 oranında koyun serumu ile kalp örnek engelleyin. </li><li> Β-katenin antikor 1:200 seyreltme ve mef2 spesifik antikor 1:200 seyreltme gibi birincil antikorlar, kalp örnek inkübe, oda sıcaklığında 1 saat süreyle% 10 oranında koyun serumu / PBST. </li><li> Oda sıcaklığında PBST üç defa (5 dk. Her biri) ile kalp örnek yıkayın. </li><li> Ikincil örnek inkübePBST 0.5 saat alexa-etiketli anti-fare ve / veya anti-tavşan antikorları 1:1000 dilüsyon gibi antikorlar,. </li><li> PBST üç kez kalp örnekleri ile yıkayın (5 dk.) Oda sıcaklığında. </li></ol> 4. Görüntüleme <ol><li> PBST çıkarın ve her bir kuyuya 10 ul montaj orta ekleyin. Çözelti berrak olana kadar birkaç dakika süreyle odasının Rock. </li><li> Montaj orta çıkarın ve mikroskop kapağı cam (24x50) ile bir slayt tüm kuyuların kapağı. </li><li> Zeiss Axioplan 2 mikroskop kullanarak balık kalp ApoTome (Carl Zeiss) ile donatılmıştır. </li></ol> 5. Temsilcisi Sonuçlar Şekil tüm zebrafish kardiyomiyositlerde membran ve çekirdekler gösteren bir görüntü bir örnek gösterilmiştir. 1. mef2c ve β-katenin antikorları, disseke 52 hpf zebrafish embriyo kalp örnekleri leke birlikte kullanılmıştır. Iken mef2c antikor lekeleri kardiyomiyositlerinin çekirdekleri, β-kateninantikor her hücre 8-10 sınırında ortaya koymaktadır. Bileşik mikroskop sahne ayarlayarak, kalp örnekleri görüntüleri aynı yönde, dış eğrilik ve kalp 11 iç eğrilik gözlem sağlayacak şekilde ayarlanabilir. Toplam kardiyomiyosit numarası, kardiyomiyosit hücre boyutu ve dairesellik sonra ölçülebilir. Şekil embriyonik bir kalbin sarkomerik yapısı ortaya başka bir örnek gösterilmiştir. 2. Biz bir disseke embriyonik kalp F59, miyozin antikor, immün yapıldı. Kalın filament çizgili bir bant, yüksek büyütme (Şekil 2) 6 açıklanacak, bütün bir embriyonik kalp myofibril ağ açıkça daha düşük büyütme açıklanacak. <img alt="Şekil 1" src="/files/ftp_upload/3510/3510fig1.jpg" /> Şekil 1 mef2 (kırmızı) ve β-katenin (yeşil) çekirdek ve outl göstermek için immünzebrafish ventrikül kardiyomiyositlerinin ines. Ölçek çubuğu = 10μm <img alt="Şekil 2" src="/files/ftp_upload/3510/3510fig2.jpg" /> Şekil 2 ya da düşük büyütme (A) veya daha yüksek büyütme (B) zebrafish ventrikül kalın filament göstermek için miyozin Immnostaining ölçekli bar = 10μm

<ol>
	<li>Shin, J. T., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Zebrafish+to+human:+modular+medical+models.">From Zebrafish to human: modular medical models.</a> Annu. Rev. Genomics Hum. Genet. 3, 311-340 (2002).</li><li>Beis, D., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+biology:+following+the+zebrafish+trend.">In vivo cell biology: following the zebrafish trend.</a> Trends. Cell. Biol. 16, 105-112 (2006).</li><li>Schoenebeck, J. J., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cardiac+development:+Advances+in+imaging+add+new+dimensions+to+the+utility+of+zebrafish+genetics.">Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics.</a> Semin. Cell. Dev. Biol. 18, 27-35 (2007).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat. Genet. 26, 216-220 (2000).</li><li>Dahme, T., Katus, H. A., Rottbauer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+the+genetic+basis+of+cardiovascular+disease.">Fishing for the genetic basis of cardiovascular disease.</a> Dis. Model Mech. 2, 18-22 (2009).</li><li>Huang, W., Zhang, R., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibrillogenesis+in+the+developing+zebrafish+heart:+A+functional+study+of+tnnt2.">Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2.</a> Dev. Biol. 331, 237-249 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book. , (2007).</li><li>Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle:+the+regulation+of+myogenesis.">Muscle: the regulation of myogenesis.</a> Curr. Opin. Genet. Dev. 4, 745-751 (1994).</li><li>Wheelock, M. J., Knudsen, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+and+associated+proteins.">Cadherins and associated proteins.</a> Vivo. 5, 505-513 (1991).</li><li>Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+hypertrophy+involves+both+myocyte+hypertrophy+and+hyperplasia+in+anemic+zebrafish.">Cardiac hypertrophy involves both myocyte hypertrophy and hyperplasia in anemic zebrafish.</a> PLoS One. 4, e6596-e6596 (2009).</li><li>Auman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+modulation+of+cardiac+form+through+regionally+confined+cell+shape+changes.">Functional modulation of cardiac form through regionally confined cell shape changes.</a> PLoS Biol. 5, e53-e53 (2007).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+zebrafish+essential+and+regulatory+myosin+light+chains+reduces+cardiac+function+through+distinct+mechanisms.">Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms.</a> Cardiovasc. Res. 79, 97-108 (2008).</li></ol>

Çıkar çatışması ilan etti.

Klasik tüm montaj immün yöntemleri ile karşılaştırıldığında, yöntem, aşağıdaki avantajları vardır. Öncelikle, çok daha güçlü floresan sinyalleri sürekli olarak geliştirilmiş penetrasyon nedeniyle elde edilebilir. Tüm montaj immün yöntemi, kalp çevresindeki yoğun cilt dokusu, tüm vücut önemli ölçüde yüksek arka planda birçok antikor penetrasyon azalır. Bu sorun özellikle büyük embriyolar için 3 gün sonrası fertilizasyon (dpf) şiddetlidir. Disseke kalpleri aksine, sadece çok ...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> tricaine </td><td> Araştırma organik </td><td> 3007T </td></tr><tr><td> Formaldehit </td><td> Polysciences </td><td> 04018 </td></tr><tr><td> Insülin enjektörü </td><td> Becton Dickinson </td><td> 329461 </td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T8532 </td></tr><tr><td> Anti-β-katenin antikor </td><td> Sigma </td><td> C7207 </td></tr><tr><td> Anti-Mef2c antikor </td><td> Santa Cruz Biotech </td><td> SC313 </td></tr><tr><td> F59 </td><td> Zfin </td><td></td></tr><tr><td> Anti-α-aktinin antikor </td><td> Sigma </td><td> A7811 </td></tr><tr><td> Anti-Ilk antikor </td><td> Hücre içi sinyal </td><td> # 3862 </td></tr><tr><td> Alexa Florlu 568 keçi anti-tavşan IgG </<html> td&gt; <td> Invitrogen </td><td> A11011 </td></tr><tr><td> Alexa Florlu 488 keçi anti-fare IgG1 </td><td> Invitrogen </td><td> A21121 </td></tr><tr><td> Montaj orta floresan </td><td> Vektör </td><td> H-1200 </td></tr><tr><td> ImmEdge kalem </td><td> Vektör </td><td> H-4000 </td></tr><tr><td> Poli-L-Lysin kaplı slaytlar </td><td> Elektron mikroskobu bilimleri </td><td> 63410-01 </td></tr><tr><td> Mikroskop kapak cam </td><td> Balıkçı </td><td> 12-543-D </td></tr><tr><td> Concaved mikroskop kızakları </td><td> Balıkçı </td><td> 7-1305-8 </td></tr><tr><td> Diseksiyon mikroskobu </td><td> Leica MZ95 </td><td></td></tr><tr><td> Bileşik mikroskop </td><td> Zeiss </td><td> Axioplan2 </td></tr><tr><td> ApoTome </td><td> Zeiss </td><td></td></tr></tbody></table> PBST: 1 x PBS % 0.5 &#39;lik Triton-X 100 ent &quot;&gt; 25X Tricaine: 400 mg Tricaine 97.9 ml GKD 2 O 2.1 mL (1M Tris pH9) PH 7.0 &#39;a ayarlayın

immunostaining of dissected zebrafish embryonic heart

Zebra balığı embriyo kalp gelişimi ve avantajlı embriyoloji ve genetik 1,2 nedeniyle insan kalp hastalıkları eğitimi için in vivo omurgalı modeli popüler bir hale gelir. Hakkında 100-200 embriyolar tek bir yetişkin balık çifti her hafta hazır. Eski utero geliştiren şeffaf embriyolar kalp kusurları 3 değerlendirmek için ideal. Herhangi bir gen ifadesi morfolino teknoloji veya RNA enjeksiyon 4 üzerinden manipüle edilebilir . Ayrıca, ileri genetik ekranlar zaten cardiogenesis 5 farklı bakış açılarını etkileyen mutant bir liste yarattı. Tüm montaj immün bu hayvan modelinde belli bir doku 6 hedeflenen protein ifade desenini ortaya çıkarmak için önemli bir tekniktir. Ancak, hücre içi veya hücre yapıları ortaya çıkarabilir yüksek çözünürlüklü görüntüleri, özellikle fiziksel locat nedeniyle olarak zor olmuşturkalp ve antikorların zayıf penetrasyon iyon. Burada, ilk kalp diseksiyon ve sonra bir mikroskop lamı yüzey boyama işlemi yapan bu darboğazları gidermek için bir yöntem sunar. Küçük kalp örneklerinin kaybını önlemek için ve çözüm kullanımını kolaylaştırmak için, bir immEdge kalem ile çizilmiş mikroskop slaytlar yüzey üzerinde bir daire içinde kalp örnekleri kısıtlı. Boyama sonra, floresan sinyalleri doğrudan bir bileşik mikroskop ile gözlemlenebilir. Embriyonik bir balık kalp sadece birkaç hücre tabakalarından oluşan antikorları için yeni bir yöntem, penetrasyon önemli ölçüde artırır. Sağlam kalpleri yüksek kaliteli görüntüler gün 2 gün 6 yaş Zebra balığı embriyolarının için çok daha alayı süre içinde elde edilebilir. Bizim metodumuz, potansiyel olarak diğer organlara ya da Zebra balığı veya diğer küçük hayvanlar disseke leke uzatılabilir.

Watch this Scientific Journal Video about Immunostaining of Dissected Zebrafish Embryonic Heart at JoVE.com

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebra balığı embriyonik kalp immün yapmak için hızlı bir şekilde açıklanmıştır. Tüm montaj immün yaklaşımı ile karşılaştırıldığında, bu yöntem, çok daha az işlem süresi içinde kalp hücre / hücre içi yapıları ortaya yüksek çözünürlüklü görüntüleri elde sağlayan antikorlar, penetrasyon önemli ölçüde artırır.

Zebra balığı Embriyonik Kalp Dissected, immün

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

immunostaining-of-dissected-zebrafish-embryonic-heart

Research

JoVE Journal

Biology

17.6K Görüntüleme.  Mayo Clinic College of Medicine. Zebra balığı embriyonik kalp immün yapmak için hızlı bir şekilde açıklanmıştır. Tüm montaj immün yaklaşımı ile karşılaştırıldığında, bu yöntem, çok daha az işlem süresi içinde kalp hücre / hücre içi yapıları ortaya yüksek çözünürlüklü görüntüleri elde sağlayan antikorlar, penetrasyon önemli ölçüde artırır.

Video: Immunostaining of Dissected Zebrafish Embryonic Heart

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Konfokal Mikroskop Zebra balığı Embriyonik Beyin Görüntüleme Canlı

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biyoloji

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Doku Hedeflenen Embriyonik Chimeras: Zebra balığı Gastrula Hücre Nakli

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Elektron Mikroskobu için embriyo Hazırlık Drosophila Embriyonik kalp tüpü

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Zebra balığı Embriyonik Göz Dokular, Mikrodisseksiyon

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

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

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

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 ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Larvaların Kalp Diseksiyon, Çocuk ve Yetişkin Zebra balığı, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

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 ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Zebra balığı Embriyonik İskelet Kası Lazer-yaralama

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

Disosiye Müstahzarlar gelen Embriyonik ve larval Zebra balığı İskelet myofibers Analizi

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Yeni Bir<em&gt; Ex vivo</emEmbriyonik Fare Kalp için&gt; Kültür Yöntemi

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...