Marie Birne, Areti Tsiola, Jaymie Estevez ve Arelys Uribe teşekkür ederiz. Bu çalışma CUNY Araştırma Vakfı ve Howard Hughes Tıp Enstitüsü Hibe Lisans Öğrencileri için Yaz Programı (SPUR) ve NIH RO3&#39;ü 41702-00-01 kısmen desteklenmiştir. Araştırma ayrıca, Queens College ve bazı deneyler Görüntüleme, Queens College&#39;da Hücresel ve Moleküler Biyoloji Core Tesisi ekipman yapıldı tarafından desteklenmiştir.

Zebra balığı 28 Queens College Hayvan Tesis 13 standart protokoller ° C ile yetiştirildik Bireysel balık çift veya grup çiftleşmelerin preformed ve Oluşan embriyolar temizlenmeli ve 28 ° C inkübatör saklanan olmalıdır. Balık sağlık günlük muayene edilmelidir. 5 gün sonrası fertilizasyon (dpf), 10-15 balık yoğunlukta balık tanklarının içine ayrılmış olmalıdır ve balık tesisi kreş girdi. 15, 30, 45, 60, 90, 180 dpf&#39;e: Burada sunulan protokolü için zebrafish ergin aşamalardan larva toplanmıştır. 1. Zebra balığı konu toplama ve fiksasyon <ol><li> Uyutmak için% 0.2 Tricaine net ve yerinde kullanarak balık habitatı tankı çıkarın. </li><li> Euthanize için 15 dakika boyunca buzlu suda anestezi balık yerleştirin. </li><li> 1x taze% 4 paraformaldehid (PFA) Fosfat-tamponlu salin (PBS). </li><li> 4 gece boyunca oda sıcaklığında 1-2 saat süreyle% 4 PFA toplanan balık inkübe ° C parafilm sarılmış bir petri. Bu balık düz tutar ve daha sonra diseksiyon çok daha kolay hale getirir. </li><li> Fiksasyon sonra, Tween (PBT) ile iki kez PBS içinde yıkayın. Balık 10 gün, 4 ° C&#39;de PBT tutmak olabilir. </li></ol> 2. Ölçüm balık ve diseksiyon <ol><li> Petri yarısını içine yerleştirin tek balık, taze PBS ile dolu ve sağ tarafta yatıyordu. </li><li> Burnu baz kuyruk (kuyruk sapı) balık Tedbir uzunluğu, kuyruk yüzgeçleri, dijital mm kaliper (Şekil 1) kullanarak da dahil olmak üzere değil. Bu standart uzunluk (SL). </li><li> Her balık bir rakam / harf kombinasyonu, daha sonraki analiz için izlenebilir için tek bir balık ve çıkan disseke kalp izin atanabilir. Ebeveyn stok numarası, fiksasyon yaşı, SL ölçümleri de dahil olmak üzere veriler, bir elektronik tablo içine girildi ve bu etiketlerin göre düzenlenir. </li><li> Diseksiyonu, etiket PCR tüp atanan takip numarası ile şeritler ve PBT veya daha fazla analiz için uygun başka bir çözüm ile doldurmadan önce. </li><li> Orient petri balık ventral yüzü yukarı, gözleri ve solungaçları (Şekil 2) arasında kafa tutan forseps ile vücut stabilize. </li></ol> Küçük balık (12mm SL daha küçük): <ol start="4"><li> a) bir iğne tutucu keskin forseps, ya da bir mikro iğne kullanarak, kalbin ortaya vücudun göğüs kasları ve yüzgeçleri çıkarın. Diseksiyon sırasında forseps sürekli hareket balık taşımak PBT akımları neden olabilir. Bu dolayısıyla bir mikro-iğne, göğüs kasları ve yüzgeçleri çıkarın ve vücut boşluğu açmak için kullanılabilir küçük balık, özellikle sorunlu. </li><li> a) yavaşça avlunun altındaki boşluktan kalp oymak için forseps kullanabilir. Alternatif olarak, bazı balık hafifçe soğanlı çekerek ve kalbin geri kalanı takip edecek kalp kaldırmak mümkün olabilir. Kolayca zarar görebilir, bu tekniği kullanarak, atrium yerini dikkatli olun. , Kalp, ince ve sonra silinebilir ile gelir ekstra bir doku vardır. </li><li> balık Tg (myl7 gibi bir floresan kalp işareti içeriyorsa: a) GFP) twu34 diseksiyon için floresan kapsamı ve floresans (Şekil 3) ile kalp kaldırma doğrulamak. </li><li> a) Bir kere kalp boşluğuna kaldırılır, kalbin dışarıdan ekstra kardiyak olmayan doku ve epikardiyal astar kaldırmak için bir kalp ve bir mikro-iğne tutmak için forseps kullanabilir. </li></ol> Büyük balık (12mm SL daha fazla): <ol start="4"><li> enine kesilmiş solungaçları üzerinden 2 - 3 ön göbek enine kesme keserek bağlayan ventral tarafta sagital kesme (Şekil 4): 1 b) ele mikro-makas yay kullanarak, 1mm derin daha az üç insizyon olun. </li><li> b) keskin forseps kullanarak vücut boşluğunda açık ve perikard gümüş doku (Şekil 5A) ortaya çıkarmak için vücudun göğüs kasları ve yüzgeçleri kaldırmak. Bu perikardiyal doku çıkarın ve kalp görünür hale gelecektir (Şekil 5B). </li><li> b) kalp üstün bulunan soğanlı arteriozus, bağlı arter kesmek için mikro-makas kullanın. Bu arter kesilir sonra, avlunun altındaki forseps ipuçları koymak, kalp boşluğuna oymak için forseps kullanabilir. , Kalp, ince ve daha sonra silinebilir ile gelir ekstra bir doku vardır. </li><li> b) kalp boşluğuna kaldırıldıktan sonra, iki kalp tutmak için forseps kullanabilir ve kalp tutmak için ekstra doku, ya da forseps bir çift kaldırmak ve bir mikro iğne ekstra, non-kardiyak doku kaldırmak için. Doku hafifçe kazıma tarafından istenirse siyah pigmentasyon belirgin epikardiyal astar, kalp dışına çıkarılmalıdır. </li></ol> 3. Fotoğraf: kupa <ol><li> 23g / L besin agar petri hazırlayın ve PBS ile pekiştirdi agar kapağı. </li><li> Forseps kullanarak, kalp, fotoğraf çekimi için istenilen yönde dinlenmesini sağlar agar iyi olun. </li><li> Remove soğanlı ucu forseps ile sıkıca tutarak PCR tüp kalp. Agar çanak kalp yerleştirin. </li><li> Orient fotoğraflamak için agar kalbinde. Örnek olası tüm yönelimleri (Şekil 6) her bir resim sonra döndürülebilir. </li><li> Kalbini çekmek için üstten hafif ve kısa pozlama süresi kullanın. , En iyi fotoğraf elde etmek için gerekli poz kullanarak ışık kalitesi için ayarlamalar yapın. </li></ol> 4. Temsilcisi Sonuçlar: Şekil 6 &#39;da birden fazla yönelimleri, iyi disseke temiz kalp bir örnek gösterilmiştir. Her kalp genel morfolojisi bazı farklılıklar var, ancak kalp sağlam bir ventrikül, atrium ve soğanlı arteriozus içeren, tam olmalıdır. Genel morfolojisi bu varyasyon bazı örnekler Şekil 7 de gösterilmiştir. Siyah benekli epikardiyal kalp dokusu da kaldırılır. <img src="/files/ftp_upload/3165/3165fig1.jpg" alt="Şekil 1"/> Şekil 1: burnu bir dijital mm kaliper kullanarak kaudal sapı balık ölçün. <img src="/files/ftp_upload/3165/3165fig2.jpg" alt="Şekil 2"/> Şekil 2, gözleri ve solungaçları arasında forseps ile balık tutun . <img src="/files/ftp_upload/3165/3165fig3.jpg" alt="Şekil 3"/> Şekil 3 Tg olarak kardiyak spesifik floresan transgenik işaretleyici (myl7 GFP) Burada gösterilen twu34, küçük balık disseke kalpleri görselleştirme yardımcı olabilir. <img src="/files/ftp_upload/3165/3165fig4.jpg" alt="Şekil 4"/> Şekil 4 pektoral kaslar ve cilt kaldırmak için üç insizyon: 1 - 2 ile solungaçları enine kesilmiş ön göbek enine kesilmiş 3 - hem de keser bağlayan ventral tarafta sagital kesim. <img src="/files/ftp_upload/3165/3165fig5.jpg" alt="Şekil 5"/> Soğanlı arteriosus (ba), ventrikül (v) ve atrium (a): Şekil 5 Siyah harfler kalp odaları etiket. A-üst vücut boşluğu perikard gümüşi doku göstermek için açık. B-sonra perikard kaldırarak, kalp görünür ve kolayca temizlenebilir. <img src="/files/ftp_upload/3165/3165fig6.jpg" alt="Şekil 6"/> Şekil 6 farklı yönelimleri kalp gösterilmiştir. : Siyah harfler kalp odaları soğanlı arteriosus (ba), ventrikül (v) ve atrium (a) etiket. Anterior (A), (P), sırt, arka (D), ventral (V), sol (L) ve sağ (R): Gri oklar yeri ve yönü, kalbin vücudun normal konuma referans gösterir. Ölçek çubuğu 400μm gösterir. Kalp A-ventral B-kalp dorsal C-lateral sol tarafında Sağ tarafında D-lateral <img src="/files/ftp_upload/3165/3165fig7.jpg" alt="Şekil 7"/> Şekil 7 farklı boyutlarda kalplerin Morfoloji gösterilmiştir. : Siyah harfler kalp odaları soğanlı arteriosus (ba), ventrikül (v) ve atrium (a) etiket. Ölçek çubuğu 400 mikron belirtti. 12mm daha az SL AC-Hearts formu balık 12mm daha büyük SL ile DF-Hearts formu balık

<ol>
	<li>Ahmad, N., Long, S., Rebagliati, 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+southpaw+joins+the+roster:+the+role+of+the+zebrafish+nodal-related+gene+southpaw+in+cardiac+LR+asymmetry.">A southpaw joins the roster: the role of the zebrafish nodal-related gene southpaw in cardiac LR asymmetry.</a> Trends Cardiovasc Med. 14, 43-49 (2004).</li><li>Armstrong, E. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+endothelial+cell+signaling+and+differentiation.">Heart valve development: endothelial cell signaling and differentiation.</a> Circ Res. 95, 459-470 (2004).</li><li>Bakkers, J., Verhoeven, M. 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Çıkar çatışması ilan etti.

Post-embriyonik balık zebrafish kalp diseksiyonu için bu yöntemleri hasarsız bir kalp tümüyle kaldırılmasını sağlar. Bu teknikte basittir ve dış kesimler için kalbin zarar görmesini önlemek kılavuzları sağlarken, sürekli bir yandan küçük balıklar için önemlidir. Diseksiyonu sırasında bir önemli adım kalp bu kese içinde doğrudan gümüş perikard belirlemektir. Perikard görselleştirme vücut boşluğunda bulma ve sonuçta kalp formunu kaldırarak önemli bir ...

<table border="1"><tbody><tr><th> Malzeme </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Paraformaldehit </td><td> EMD </td><td> PX0055 </td><td> PBS içinde .04g/mL </td></tr><tr><td> Forseps # 55 </td><td> FST </td><td> 11295-51 </td><td></td></tr><tr><td> Mikro-makas </td><td> FST </td><td> 15005-08 </td><td></td></tr><tr><td> Pin tutucu </td><td> FST </td><td> 26016-12 </td><td></td></tr><tr><td> Mikro-needles </td><td> FST </td><td> 10130-10 </td><td></td></tr><tr><td> Nutrient Agar </td><td> BBL </td><td> 11472 </td><td> Distile H 2 O 23g / L </td></tr><tr><td> Petri kutularına </td><td> BD Falcon </td><td> 351029 </td><td></td></tr><tr><td> PCR 8-tüp şeritler </td><td> ABD Bilimsel </td><td> 1402-2500 </td><td></td></tr><tr><td> Dijital Kaliper </td><td> Fisher Scientific </td><td> 14-648-17 </td><td></td></tr></tbody></table>

heart dissection in larval, juvenile and adult zebrafish, danio rerio

Zebra balığı çalışma (son değerlendirmeleri görmek 1-6) embriyonik kalp gelişimi için yararlı ve pratik bir model organizma haline gelmiştir, ancak 7-10 sınırlı kalmıştır yetişkin kalp geliştirilmesi yoluyla post-embriyonik inceleyerek işe . Olgunlaşan ve yaşlanma kalp değişen morfoloji incelenmesi, genç ve yetişkin kalpleri evreleme ve izole etmek için uygun teknikler eksikliği ile sınırlıdır. Balığın ömrü boyunca kalp gelişimini analiz etmek için, çeşitli aşamalarda zebrafish kalpleri incelemek ve onları daha fazla analiz için 11 fotoğraf. Kalbin morfolojik özellikleri kolayca sayısal ve bireysel kalpleri daha bir dizi standart yöntemler ile analiz edilebilir. Zebra balığı değişken oranlı büyüme ve olgunlaşma, dolayısıyla balık olgunlaşma bir göstergesi olarak post-fiksasyon, fotoğraf ve ölçü balık uzunluğu, yaş daha balık boyutu ile daha fazla ilişkili. Bu protokol ile 30mm ve VL 1mm SL veya daha büyük, yetişkin 100μm ventrikül uzunluğu (VL) kalplerini larva 3.5mm standart uzunluğu (SL) arasında değişen iki ayrı zebrafish için, boyut bağımlı diseksiyon teknikleri, açıklar. Larva ve ergin balıklar oldukça farklı bir vücut ve organ morfolojisi vardır. Larva sadece daha küçük değil, daha az pigment ve her organ görsel olarak tespit etmek çok zordur. Bu nedenle, biz farklı diseksiyon teknikleri kullanır. Euthanization sonra doğrudan disseke kalpleri kulakçıklar gibi çok gevşek ve balon ile aşağıdaki fiksasyon kaldırıldı kalpleri ile karşılaştırıldığında, daha değişken bir morfoloji olduğunu keşfettiği gibi, pre-diseksiyonu fiksasyon yöntemleri kullanılır. Balık, in vivo morfolojisi ve oda pozisyonlar (veriler gösterilmemiştir) korumak diseksiyon önce sabit . Buna ek olarak, gösteri amaçlı, kalp (miyokard) belirli GFP transgenik Tg (myl7 GFP) yararlanmak için bize tüm kalp görüntülemenizi sağlar ve geliştirme erken aşamalarında özellikle yararlıdır twu34 (12), kalp morfolojisi çevre dokulara daha az belirgindir. Kalp Diseksiyon in situ hibridizasyon, immunohistokimya, RNA ekstraksiyonu veya diğer post-embriyonik zebrafish kolay analitik yöntemler kullanarak kalp gelişme ve olgunlaşma altında yatan hücre ve moleküler biyoloji daha fazla analiz yapar. Bu protokol, kalp gelişim olgunlaşma ve yaşlanma çalışma için değerli bir teknik sağlayacaktır.

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

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

Birden fazla gelişim evreleri zebrafish kalp diseksiyonu ve izolasyon için açık, standart bir yöntem açıklanmıştır. Annotation ve miktar teknikler de tartışılmaktadır.

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 ...

heart-dissection-in-larval-juvenile-and-adult-zebrafish-danio-rerio

Research

JoVE Journal

Biology

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

21.3K Görüntüleme.  Queens College, City University of New York. Birden fazla gelişim evreleri zebrafish kalp diseksiyonu ve izolasyon için açık, standart bir yöntem açıklanmıştır. Annotation ve miktar teknikler de tartışılmaktadır.

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

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Drosophila melanogaster Larvaların MSS diseksiyonu

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Biyoloji

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila Larvaların NMJ Diseksiyon

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Yetişkin Zebra balığı İç Kulak Duyu Epiteli Brüt ve Güzel Diseksiyon ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Yetişkin Zebra balığı Organları diseksiyonu

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Diseksiyon ve boyanması Drosophila Larvaların Yumurtalıkların

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Yetişkin Zebra balığı Böbrek diseksiyonu

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos in vivo Elektroporasyon Yetişkin Zebra balığı Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

Bir Zebra balığı Düzenli Bakım ve Onarım (<em&gt; Br.rerio</em&gt;) Laboratuvar: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Br.rerio kullanarak Morbid İnsan Genomu İn Vivo Modelleme

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Yetişkin Zebra balığı Gavaging

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...