FIPE / HCPA - Fundo de Incentivo bir Pesquisa e Eventos Pelerin - <a href="http://www.capes.gov.br/">Coordenação de Aperfeiçoamento de pessoal de Nivel Üstün</a>

1. Protokol Metni <ol><li> Zebrafish kan toplama önce, anestezi su hazırlamak için gereklidir. 500 ml kapasiteli bir konteyner içine akvaryum su ~ 200 ml dökün. Buz yongaları ~ 200 g ekleyin. 4 ° C sıcaklıkta yaklaşık olmalıdır Buz yongaları eriyik gibi, 4 ° C civarındaki sabit bir sıcaklıkta muhafaza etmek daha buz yongaları eklemek için gerekli olacaktır </li><li> Anestezi su hazır olduğunda, kan toplama için gerekli malzemeleri hazırlar. Bir P20 pipet düşük tutma ucu koyun ve kolayca erişilebilir pipet bırakın. Pipet bulaşma kaynakları temasa izin vermeyin. </li><li> Kuru gazlı bez parçası ile Petri örtün. Bir çelik bıçak ve gazlı bez başka bir parça kolay erişilebilen bir yere konulmalıdır. </li><li> Plastik borular için uyarlanmış bir santrifüj gerekli olacaktır. </li><li> Söz konusu malzeme hazırlandığı zaman, anestetize wit olarak birinci zebrafish yakalamakha balık ağı ve anestezi için hazırlanmış olan suya bırakın. Zebrafish soğutulmuş su içinde 3-6 s balık bağlı olarak anestezi uygulanmış olması gerekir. Artık dış uyaranlara yanıt verene kadar soğuk suda balık tutun. </li><li> Balık ağı kullanarak, tül kuyruk dışı bırakarak, gazlı bez hazırlanmış bir parçası üzerinde anestezi balığı yerleştirin. Sadece kuyruğunu dışarıda bırakarak balığın baş ve vücut üzerinde gazlı bez katlayın. Petri tabağına gazlı bez ile kaplı balık koyun. </li><li> Sadece anal yüzgeç ve kuyruk yüzgeci arasında çapraz bir kesi yapmak için çelik bıçak kullanın. Kan dışarı çıkmaya başlayacaktır. Bu noktada, hızlı bir şekilde çalışması için gereklidir. </li><li> Yavaşça P20 pipet (düşük tutma ucu ile ön yüklü) ile çıkan kanın talip. Toplanabilir kan miktarı balık boyutuna ve ne doğru anestetize olduğu ölçüde değişir. Bu, genellikle 5 ile 20 ul kadar değişir. Kan c dururdışarı oming, yavaşça bir tüp içine aspire kan aktarın. </li><li> Hemoliz önlemek için, bunun santrifüj yerleştirilir kadar kan ile tüp herhangi bir sert hareketler yapmadan, çok dikkatle ele alınması önemlidir. </li><li> Hemoliz önlemek için, kan örneği kan toplama 10 dakika içinde santrifüj temin edilmesi de önemlidir. </li><li> Eğer gerekirse, bunun bir havuz verme, birden fazla hayvandan kan birleştirmek mümkündür. Pooled örnekleri ilk balık ve santrifüj kan toplama arasındaki gecikme 10 dakika aşmadığı sürece iyi çalışır. </li><li> Kan toplama yapıldığında, 0.5 g (Eppendorf Santrifüj 5415D) 10 dakika boyunca kan santrifüjlenir. </li><li> Santrifüjleme takiben, serum borusunun üst tabaka yer almaktadır. Bir pipet ile, her iki katmana da bölünmüştür ve istikrarlı tutarken sadece serum almak için emin, serum aspire. </li><li> Yeni bir mikrotüp içine serum transferi ve bubiyokimyasal analizlerde kullanılmak üzere hazırdır. Bu biyokimyasal analizler başlamak için beklerken serum buz saklanabilir. </li><li> Serum hemen kullanılmayacak ise, yaklaşık 3 ay -18 ° C kadar dondurulabilir. </li></ol> 2. Temsilcisi Sonuçlar Daha önce açıklanan teknikler (Tablo 2) bile 4 kat daha fazla kan neyi temsil ettiğini her balıktan tam kan 5 ila 20 ul toplamak mümkün olmuştur. Total kolesterol, HDL-kolesterol, LDL-kolesterol ve trigliserid biyokimyasal analizler bu tekniği kullanarak kan alımından sonra yapıldı. Kan toplama gıda alımı girişimi önlemek için önce her iki cinsiyette balık iki grup 24 saat aç bırakıldı. Analizleri total kolesterol ve trigliserid analizleri için küçük ölçekli kolorimetrik testler (Labtest Diagnostica SA, Brezilya) ile yapıldı, serum 3 ul kullanılmıştır. LDL-kolesterol ve HDL-kolesterol içinanalizi, 4 ul ve 10 ul serum sırasıyla kullanılmıştır. Bu analizler, örnek başına 10 zebrabalıkları havuzlanmış örnekleri üzerinde yapılmıştır. Serum lipidik düzeyleri kendi yumurta ve bir alt kapalı akvaryum, 2 haftalık bir deneme süresi için kendi yumurtalarını erişimi olmadığını, bu erişilebilir balıklar arasında karşılaştırıldı. Serum analizleri, serum total kolesterol düzeyleri (yumurta 362 ± 42 mg / dL ve yumurta 357 olmadan ± 13 mg / dL), HDL-kolesterol (yumurta 91,22 ± 1.79 mg / dL ve yumurta 72.14 olmadan ± 2.89 mg / dL) ve (yumurta 55,68 ± 10.88 mg / dL ve yumurta 44.18 ± 9.84 mg / dL olmadan) LDL-kolesterol gruplar arasında anlamlı farklılık yoktu. Ancak, trigliserid düzeyleri (p = 0.03 yumurta 457 ± 25 mg / dL) ile kontrol grubuna göre (yumurta 292 ± 64 mg / dL olmadan) deney grubunda anlamlı olarak daha düşüktü. <table border="1" ><tbody><tr><td> &amp; Nbsp; </td><td> Yumurta erişim </td><td> Yumurta erişimi olmayan </td></tr><tr><td> Total Kolesterol (mg / dL) </td><td> 362,82 ± 73,11 </td><td> 357,69 ± 23.08 </td></tr><tr><td> LDL - kolesterol (mg / dL) </td><td> 55,69 ± 18,84 </td><td> 44.19 ± 17.05 </td></tr><tr><td> HDL - kolesterol (mg / dL) </td><td> 91,23 ± 3.11 </td><td> 72.14 ± 5.01 </td></tr><tr><td> Trigliserid (mg / dL) </td><td> 457,64 ± 43.78 * </td><td> 292,36 ± 111,28 </td></tr></tbody></table> Tablo 1. Her iki çalışma grupları için kolesterol ve trigliserid düzeyleri Seric (yumurta erişimi olan ve yumurta erişim olmadan) ortalama ± standart sapma olarak ifade edilmiştir. * Istatistiksel olarak anlamlı (p = 0.03). Student t testi. <table border="1"><tbody><tr><td> Aut<html> hors </td><td> Kesi yeri </td><td> Hasat yöntemi </td><td> Anestezi </td><td> Toplanan kan miktarı </td></tr><tr><td> Jagadeeswaran et. ark., 1999 Murtha ve ark., 2003 </td><td> Dorsal yüzgeç Micro diseksiyon posterior </td><td> Mikropipet </td><td> Bahsedilmeyen Soğuk su içinde% 3 MS222 </td><td> 1, bir 5 ul 5, 10 ul </td></tr><tr><td> Eames ve ark., 2010 </td><td> Işten çıkarma </td><td> Mikro kılcal tüp </td><td> MS222% 0,02 28 ° C su </td><td> 5, 10 ul </td></tr><tr><td> Mevcut Çalışma </td><td> Anal yüzgeç ve kuyruk yüzgeci arasında Kesi </td><td> Mikropipet ve düşük tutma ipuçları </td><td> Su ve buz yongaları </td><td> 5, 20 ul </td></tr></tbody></table>daha önce açıklanan kan toplama teknikleri ve bu çalışmada adresinde açıklanan biri arasında e_content &quot;&gt; Tablo 2. karşılaştırılması.

<ol>
	<li>Schneider, A. C. R., dos Santo, J. L., Porawski, M., Schaefer, P. G., Maurer, R. L., Matte, U., da Silveira, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementa&#231;&#227;o+de+um+novo+modelo+de+experimenta&#231;&#227;o+animal+Zebrafish.">Implementa&#231;&#227;o de um novo modelo de experimenta&#231;&#227;o animal Zebrafish.</a> Rev. HCPA. 29, 100-103 (2009).</li><li>Briggs, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish:+a+new+model+organism+for+integrative+physiology.">The zebrafish: a new model organism for integrative physiology.</a> Am. J. Physiol. Regulatory Integrative Comp. Physiol. 282, 3-9 (2002).</li><li>Menke, A. L., Sptsbergen, J. M., Wolterbeek, A. P. M., Woutersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+anatomy+and+histology+of+adult+Zebrafish.">Normal anatomy and histology of adult Zebrafish.</a> Toxicologic Pathology. 000, 1-16 (2011).</li><li>Murtha, J. M., Qi, W., Keller, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematologic+and+serum+biochemical+values+for+Zebrafish.">Hematologic and serum biochemical values for Zebrafish.</a> Comp. Med. 53, 37-41 (2003).</li><li>Jagadeeswaran, P., Sheehan, J. P., Craig, F. E., Troyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+zebrafish+thrombocytes.">Identification and characterization of zebrafish thrombocytes.</a> Br. J. Haematol. 107, 731-738 (1999).</li><li>Jagadeeswaran, P., Sheehan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blood+coagulation+in+the+zebrafish.">Analysis of blood coagulation in the zebrafish.</a> Blood Cells Mol. Dis. 25, 239-249 (1999).</li><li>Eames, S. C., Philipson, L. H., Prince, V. E., Kinkel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sugar+measurement+in+zebrafish+reveals+dynamics+of+glucose+homeostasis.">Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis.</a> Zebrafish. 7, 205-213 (2010).</li><li>Kilpatrick, E. S., Rumley, A. G., Rumley, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+haemolysis+on+blood+glucose+meter+measurement.">The effect of haemolysis on blood glucose meter measurement.</a> Diabet. Med. 12, 341-343 (1995).</li><li>Gupta, T., Mullins, 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=Dissection+of+Organs+from+the+Adult+Zebrafish.">Dissection of Organs from the Adult Zebrafish.</a> J. Vis. Exp. (37), e1717 (2010).</li><li>Kinkel, M. D., Eames, S. C., Philipson, L. H., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+Injection+into+Adult+Zebrafish.">Intraperitoneal Injection into Adult Zebrafish.</a> J. Vis. Exp. (42), e2126 (2010).</li><li>Pugach, E. K., Li, P., White, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+Injection+in+Adult+Zebrafish.">Retro-orbital Injection in Adult Zebrafish.</a> J. Vis. Exp. (34), e1645 (2009).</li></ol>

Çıkar çatışması ilan etti.

Bu kağıt zebrabalıkları deneylerde daha fazla kan ve serum analiz sağlayan basit bir teknik sunuyor. Bu teknik, kan parametre veri gerektiren gelecekte zebrabalıkları hematolojik çalışmalara katkıda bulunma potansiyeline sahiptir. Ayrıca, bir deneysel model olarak zebrafish daha fazla uygulamalar için izin vermek gerekir. Bu teknik, özel bir beceri ya da kesin bir tekniğin uygulanmasını gerektirmez. Dahası, bu sayede, biyolojik malzemenin gerekli miktarda elde etmek için d...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Düşük tutma ipuçları </td><td> Applied Biosystems </td><td> 022493020 </td><td></td></tr><tr><td> Eppendorf Centrifuge 5415D </td><td> Eppendorf </td><td> Durduruldu </td><td></td></tr></tbody></table>

blood collection for biochemical analysis in adult zebrafish

Zebrafish çok insan hastalığı çalışmaları için bir hayvan modeli olarak kullanılmıştır. Bu moleküler olaylar ve tedavi edici stratejilerin çalışmaları yanı sıra bazı patolojiler 1 fizyolojik mekanizmaların değerlendirilmesi için güçlü bir preklinik bir platform olarak hizmet verebilir. Araştırmacılar zebrabalıkları sistemlerinin araştırılması izin vermek için gerekli temel teknikleri 3 eksik olduğu sonucuna yol açabilir organ ve sistemleri 2, yetişkin zebrabalıkları fizyolojisi ile ilgili nispeten az sayıda yayın vardır. Zebra balığı hematolojik biyokimyasal değerler ilk önce 1999 yılında Jagadeeswaran ve arkadaşları tarafından tanımlanan bir kan toplama tekniği istihdam Murtha ve arkadaşları 4 tarafından 2003 yılında bildirilmiştir. Kısaca, kan uzunluğu, yaklaşık 0.3 cm dorsal aorta 5 bölgede, yanal bir kesi mikropipet ucu ile toplanmıştır. Söz dakikalık boyutlar, bu yüzdenBir çok yetenekli uygulayıcısı gerektiren yüksek hassasiyetli bir tekniktir. Aynı teknik aynı yıl 6 başka yayın aynı grup kullanılmıştır. 2010 yılında, Eames ve arkadaşları Zebra balığı 7 tam kan glikoz düzeyleri değerlendirildi. Onlar makasla decapitations performans ve daha sonra göğüs eklem içine bir heparinize microcapillary toplama tüpü takılarak kan erişim kazandı. Onlar iş Kilpatrick ve ark göre uygun bir depolama sıcaklığı ile çözüldü hemoliz zorluklar söz. 8. Laboratuvarımızda Jagadeeswaran tekniğini kullanmaya çalışırken, biz toplama başlamış olabilir önce kaybolmuş kan önemli miktarda izin vermeme gibi kesin doğru yerde kesi yapmak için zor olduğunu bulundu. Son zamanlarda, Gupta ve ark. 9 yetişkin zebrabalıkları organları, Kinkle ark incelemek anlattı. 10 intraperiton nasıl gerçekleştirileceği açıklananeal enjeksiyonları ve Pugach ve ark. 11 retroorbital enjeksiyonları nasıl gerçekleştirileceği açıklanmıştır. Ancak, daha fazla iş daha tam zebrabalıkları araştırma için temel teknikler araştırmaya ihtiyaç vardır. Zebra balığı sayısının küçük bir deneysel model olarak kullanan araştırmacılar için büyük güçlükler. Ayrıca ölçek küçüklüğü, bu verilen, basit teknikleri araştırmacılar zebrabalıkları modelin avantajlarını keşfetmek sağlamak amacıyla geliştirilmiş olması önemlidir.

Watch this Scientific Journal Video about Blood Collection for Biochemical Analysis in Adult Zebrafish at JoVE.com

Blood Collection for Biochemical Analysis in Adult Zebrafish

Bu kağıt Zebra balığı dorsal aortadan kan toplanması için bir teknik sunuyor. Ayrıca bu tür kolesterol ve trigliserid düzeylerini belirlemek için testler gibi biyokimyasal analizlerde kullanılmak üzere serum elde etmek için yönergeler sağlar.

Yetişkin Zebra balığı Biyokimyasal Analiz için Kan Alma

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

blood-collection-for-biochemical-analysis-in-adult-zebrafish

Research

JoVE Journal

Biology

36.3K Görüntüleme.  Centro de Pesquisa Experimental Laboratório de Hepatologia e Gastroenterologia Experimental. Bu kağıt Zebra balığı dorsal aortadan kan toplanması için bir teknik sunuyor. Ayrıca bu tür kolesterol ve trigliserid düzeylerini belirlemek için testler gibi biyokimyasal analizlerde kullanılmak üzere serum elde etmek için yönergeler sağlar.

Video: Blood Collection for Biochemical Analysis in Adult Zebrafish

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebra balığı Beyin Ventrikül Enjeksiyon

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Biyoloji

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Yetişkin Zebra balığı retro-orbital Enjeksiyon

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

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 ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Yenileyici Yetişkin Zebra balığı Tail Fin içine Morpholinos in vivo olarak Elektroporasyon

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Glikojenin Biyokimyasal titrasyonu<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

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 ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Yetişkin Zebra balığı Lateral Hat Rejenerasyon için bir Deneyi

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

Yetişkin Zebra balığı Böbrek Nefron Kompozisyon ve Fonksiyon Analizi

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Yetişkin Zebra balığı Kan Testleri için Tekrarlanan Kan Alma

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

Yetişkin Zebra balığı Biyokimyasal Analiz için Kan Alma

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