S843: LI1 ve Tg (EGFP kdrl): Biz Tg (GFP wt1b) hediye Prof. Christoph Englert ve Prof. Didier Stainier teşekkür suşu, sırasıyla, Tg (kdrl: mCherry) sağlanması Tayvan balığı Çekirdek Tesis ci5. Bu çalışma 102-, Bilim ve Teknoloji (96-2628-B-029-002-My3, 101-2313-B-029-001, 102-2628-B-029-002-My3 Tayvan Bakanlığı hibe ile desteklenmiştir 2321-B-400-018).

Etik Beyanı: zebrabalıkları tüm deneysel prosedürleri Tunghai Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi (KİK Onay NO 101-12.) Ve onaylı kurallarına uygun olarak gerçekleştirdi. 3β-Hsd enzimatik Aktivite Boyama için 1. Stok Çözümleri <ol><li> trans-dehidroepiandrosteron [dimetil sülfoksit (DMSO) içinde 10 mg / ml] hazırlayın. </li><li> β-nikotinamid adenin dinükleotid hidrat (0.1 M fosfat tampon maddesi içinde 1.2 mg / ml, pH 7.2) hazırlayın. </li><li> Nikotinamid hazırlanması (B3 vitamini, 50 mg / ml H2O). </li><li> 4-nitro mavi tetrazolyum (50 mg / ml% 70 dimetilformamid) hazırlayın. </li><li> -20 ° C&#39;de mikrofüj tüpleri ve mağaza içine kısım tüm bu bileşenler. NOT: bizim deneyim, tekrarlanan donma ve tüm yukarıda belirtilen çözümlerin hacimde çözülme ise 3β-Hsd etkinlik boyama yoğunluğunu etkilemez. </li></ol> 2. Wdelik montaj 3β-Hsd Etkinlik Boyama Not: 3β-HSD &#39;enzimatik aktivitesi erken 28 saat sonra döllenme (HPF) halinde kültürlendiğinde hsd3b1 2,7 mRNA ifadesinin başlangıcı ile tutarlıdır 28.5 ° C, zebra balığı embriyolar tespit edilebilir. Bu çalışmada, tüm montaj 3β-HSD &#39;aktivitesi boyama protokolü daha önce tarif edilen yönteme 8 dayanır ve 7 gün sonra gübreleme 9 gelişmekte zebrabalıkları başarılı olduğu saptanmıştır. Interrenal steroidojenik doku damar mikro-analizi için, 3β-HSD &#39;aktivitesi boyama tüm montaj gibi Tg olarak endotel spesifik floresan ifade eden transgenik kuşaklar (kdrl: EGFP) uygulanması gerekir: ci5 15 14 ve TG (MCherry kdrl) s843 . Zebra balığı böbrek gelişimini göz önünde bulundurarak interrenal steroidogenik doku analiz etmek, 3β-Hsd etkinlik boyama başvurunun olabilirLI1 balık 16 glomerülleri ve Pronephric tübüller oluşumu tarif edilmektedir: Tg (GFP wt1b) ED. <ol><li> Pigment oluşumunu inhibe etmek için, 12 ve 24 HPF arasındaki aşamasından, yumurta suda% 0.03 feniltiyoüre bölgesindeki (deiyonize su içinde 0.06 mg / L resif tuzu) onları kuluçka geliştirme zebrafish tedavi edin. </li><li> gece boyunca, oda sıcaklığında (RT) ya da (B),% 2 PFAT de 1 saat süre ile,% 0.1 Tween 20 (PFAT) ile desteklenmiş fosfat tamponlu tuz (PBS) içinde (A)% 4 paraformaldehit (PFA) dechorionated embriyolar veya larvaları saptamak 4 ° C ve oda sıcaklığında 4 saat ve ardından 4 ° C&#39;de 2 saat için. Dikkat: PFA Solunduğunda ve cilt ile temasında toksiktir. Bu cilt, mukoza zarları, gözler ve üst solunum yollarında yıkıcıdır. </li><li> PBS oda sıcaklığında,% 0.1 Tween 20 (PBST) ile desteklenmiş olan 10 dakika için embriyo 4x yıkayın. NOT: yıkama solüsyonları değiştirirken örnekleri kuru değil için çok önemlidir. </li><li> Taze sta hazırlamakreaksiyonlar daha önce ining çözeltisi (Tablo 1). Hazırlamak girdap ve ayrı tüplerde santrifüj parçaları A ve B. NOT: Düz tek tüp içine tüm bileşenleri ekleme şiddetli yağış neden olur. </li><li> , B kısmının tamamı çözeltiye A kısmının tüm solüsyonu ilave boyama solüsyonu hazırlamak için iyice karıştırın ve hemen sabit ve yıkandı embriyoları içeren her bir mikrofüj tüpüne 1 mi dağıtın. </li><li> rotator veya oda sıcaklığında nazik çalkalama için sallanan bir platform da ışık ve yerine korumak için alüminyum folyo ile mikrofüj tüpleri sarın. </li><li> Ampirik mikroskobu ile kromojenik sinyalleri izleyerek reaksiyon süresini belirler. zamanla gelişecektir hafif yağışlar; Bununla birlikte, bu reaksiyon işlemi engel olmaz. Not: Embriyolar 4 saat 25 ° C&#39;de 28 HPF boyama sabit için interrenal doku net bir sinyal üretir. </li><li> PBST içinde 10 dakika için 4x yıkama ederek reaksiyonu durdurun. benf başka immünohistokimyasal (IHC) analizi gereklidir sonrası tamir oda sıcaklığında 60 dakika boyunca% 4 PFAT ile boyanmış embriyolar. Aksi takdirde, bir sonraki kullanıma kadar 4 ° C &#39;de yıkandı, embriyolar saklayın. </li><li> tüm montaj mikroskopi için, PBS içinde% 50 gliserol ile lekeli sonradan tespit ve yıkanmış embriyolar temizlemek bir slayt yukarı bakacak şekilde sırt tarafında onları yönlendirmek ve parlak saha aydınlatma altında dik bir mikroskop kullanılarak gözlemlemek. NOT: tavsiye edilir interrenal doku morfolojisi bir yüksek çözünürlüklü görüntü, 3β-Hsd aktivite lekeli ve deyolked embriyo analizi, düz monte yapmak için. Interrenal steroidogenik doku sağ yumurta sarısının üzerine konumlandırılmış olarak, interrenal doku ventral düz montaj analizi interrenal morfolojisi 17 doğrudan ve net gözlem sağlar. </li></ol> 3β-Hsd Etkinlik lekeli Embriyolar 3. IHK Analizi NOT: steroi vasküler mikro analiz içindogenic dokusu, bir transgenik endotel spesifik flüoresan raportör 3β-HSD &#39;aktivitesi lekeli embriyolar vibratome kesilmesi ve daha sonra IHC analizi 12,18 enine tabi tutulur. <ol><li> Post-fiksasyon <ol><li> 4 ° C &#39;de 1 saat süre ile% 1 Triton X-100 ile desteklenen% 2 PFA 3β-HSD&#39; aktivitesi-boyanmış ve yıkandı embriyolar sonrası düzeltmek ve% 1 Triton X-100 içeren PBS içinde 10 dakika boyunca 4x yıkayın (PBSTx) oda sıcaklığında karıştırıldı. </li></ol></li><li> katıştırma <ol><li> % 4, 95 ° C, 4-8 ° C de mikrofüj tüplerine kısım ve mağazada PBS içinde agaroz eritilerek agaroz erime noktası düşük hazırlayın. </li><li> 10 dakika süre ile 70 ° C &#39;de agaroz% 4 düşük erime bir kısım eritin ve sonra 47 ° C&#39;de kısım tutun. Her embriyo yerleştirme eriyik% 4 bir kalıp olarak hizmet veren bir 1.5 mi mikrofüj tüpün müstakil bir kap içinde agaroz, erime noktası düşük, ve katı kadar soğumaya bırakın. </li></ol></li><li> vibratome Kesit <ol><li> Kuru p kağıt şerit bir parça sopavibratome latform. </li><li> bir jilet ile kalıp sertleştirilmiş agaroz blok çıkarın. platformda kağıt şerit superglue uygulayın ve yukarı bakacak şekilde numunenin ön tarafı, kağıt şerit üzerine agaroz blok düzeltmek. sırasıyla, yaklaşık olarak 4 mm, üst ve alt yüzlerin her bir tarafında 8 mm olan, bir yamuk prizma şekline agaroz blok kesin. </li><li> Bir damlalık kullanılarak,% 0.5 Triton X-100 (% 0.5 PBSTx) ile desteklenmiş, soğuk PBS ile numune içeren agaroz blok durulayın. </li><li> vibratome kullanım kılavuzuna göre 100 mikron bölüme agaroz blok dilimleyin. Sürekli kurumasını önlemek için agaroz blok durulayın. Her bir dilim kesme gibi, bir 26 G iğne kullanılarak şırınga vibratome çıkarın ve soğuk% 0.5 PBSTx (500 ul / oyuk) ihtiva eden bir nokta plakaya aktarılır. </li><li> Diseksiyon mikroskobu altında kontrol ederek 3β-Hsd aktivite pozitif doku bölümleri toplayın ve bir pi ile bölümleri aktarmakSoğuk% 0.5 PBSTx (150 ul / oyuk) ihtiva eden bir 96-çukurlu plaka uç kesme (açıklık yaklaşık 1 mm iç çap) sahip Pette ucu. doku örnekleri genellikle bu aşamadan sonra agaroz blok ayırmak. </li></ol></li><li> Permeabilization ve Yıkama <ol><li> PBS içinde 10 dakika% 1 sığır serum albümini /% 1 DMSO /% 0.1 Triton X-100 (PBDTx) ile desteklenmiş dilimler, oda sıcaklığında,% 0.5 PBSTx 15 dakika PBS içinde 10 dakika ve 4 kere 6x yıkayın. </li></ol></li><li> bloke etme <ol><li> Oda sıcaklığında PBDTx 1 saat,% 2 fetal sığır serumu (FCS), dilimler inkübe edin. </li></ol></li><li> Primer antikor reaksiyonu <ol><li> Birincil antikor içeren PBDTx 1.5 gün inkübe dilimleri (örneğin, tavşan poliklonal anti-insan fibronektin ve fare anti-insan fokal yapışma kinaz) 4 ° C&#39;de ilave edildi. </li></ol></li><li> Yıkama <ol><li> Dilimler oda sıcaklığında PBDTx 10 dakika boyunca 6 kat yıkayın. </li></ol></li><li> bloke etme <ol><li> PB içinde% 2 FCS içinde 1 saat boyunca inkübe dilimleriOda sıcaklığında DTx. </li></ol></li><li> İkincil antikor reaksiyonu <ol><li> 4 ° C &#39;de ikinci bir antikor (florofor-konjuge keçi anti-tavşan veya anti-fare IgG&#39;si) ihtiva eden PBDTx 1.5 gün inkübe dilimleri. </li></ol></li><li> Yıkama <ol><li> Dilimler oda sıcaklığında PBST 10 dakika süreyle 10 oda sıcaklığında PBDTx içinde min ve 4x için 6x yıkayın. , Odaklı mikroskopik analizi için PBS içinde% 50 gliserol ile örnekleri temizlemek: NOT. </li></ol></li></ol> 4. Konfokal Analizi <ol><li> tüm montaj embriyo ve 10X / 0.5 ve 20X / 0.75 objektif lens ile donatılmış bir konfokal mikroskop ile bölümüne dikkat ediniz. </li><li> aşağıdaki gibi floresans ve 3β-HSD &#39;boyama birinin eş zamanlı saptanması için, çok kanallı ayar modu: 488 nm argon lazeri ve GFP tespiti için 505-530 nm bant geçişi filtresi; 543 nm Helyum-Neon lazer ve mCherry tespiti için 560-615 nm bant geçiş filtresi; ve 3β-Hsd boyama tespiti için ışık kanalı iletti.iğne deliği boyutu 1 Airey birimi ve çözünürlüğü 1024 x 1024 piksel olduğunu. </li><li> 3D projeksiyon olarak (6 mikron aralığı ile) z yığınları birleştirin ve kullanarak, projeksiyon ve parlak bir alan birleştirme yerleşik konfokal yazılımı. </li></ol>

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The authors have no competing financial interests to declare.

3β-HSD aktivitesinin sinyal gücü reaksiyon boyunca arttı. 3β-HSD aktivitesinin net sinyaller itibaren 28 HPF gelen aşamaları için reaksiyon 4 saat sonra tespit edildi. Bununla birlikte, reaksiyon süresi deneyi amacına bağlı olarak, ampirik belirlenmesini gerektirir. boyama gece boyunca işlem gerektirir durumlarda, hafif mavimsi arka örneklerinin geliştirme eğilimindedir. Bu sorun, 3β-HSD &#39;aktivitesi boyama önce sabitleme süresi (örneğin, oda sıcaklığında 4% PFAT 4 saat) arttırarak ...

böbreküstü bezi, hipotalamus-hipofiz-adrenal ekseninin önemli bir bileşeni, steroid salgılar ve steroid homeostazı ve strese bedensel yanıtını koordine eder. böbrek üstü bezi katekolamin sentezleyen bir bölge-spesifik bir şekilde steroid salgılayan dış korteks ve iç medulla içerir. Teleostlarda interrenal bezi, memelilerde adrenal bez karşılığıdır ve adrenal korteks ve medullada sırasıyla 1-3 fonksiyonel eşlenikleridir steroidojenik interrenal ve kromafin hücreleri, oluşmaktadır. Zebra balığı model kullanarak yapılan çalışmalar, hem steroidogenik ve kromaffin hücre soylar yüksek memelilerde 1,2 benzeyen moleküler ve hücresel mekanizmaları tarafından oluşturulan bildirmiştir. Bu nedenle, Zebra balığı, genetik bozukluklar, nöroendokrin kontrolü ve hipotalamus-hipofiz-adrenal (interrenal) ekseninin sistem biyolojisi çalışmak için bir potansiyel olarak güçlü bir modeldir. 4,5 ila 17α-hydroxypregnelolone gelen pregnenolon, 17a-hidroksiprogesteron progesteron dönüşümünü ve androstenedion katalize eder. 3β-HSD &#39;hormonal steroid, yani progesteron, glukokortikoid, mineralokortikoid, androjen ve östrojenlerin her sınıfları üreten mutantı için gereklidir. İki insan 3β-Hsd HSD3B1 izozimine ve HSD3B2 farklı şekilde 6 ifade edilir. HSD3B2 adrenal korteks ve gonadlar ifade edilir ise HSD3B1, plasenta ve periferik dokularda ifade edilmektedir. İnsan HSD3B1 ve HSD3B2 interrenal doku ve yetişkin gonadlar ifade edilen Zebra balığı hsd3b1, eş ortologlar vardır; Zebra balığı hsd3b2 olan transkript organogenez 7 önce kaybolur bir anne olarak ifade gendir. zebrabalıkları için tüm montaj 3β-HSD &#39;enzimatik aktivite deneyi protokol, Levy yöntemini değiştirerek geliştirilenMilano ve diğerleri tarafından tarif s. Sekiz Teleost türlerinin 8 donmuş bölümlerinde. Çünkü doku geçirgenliği ve gelişmekte olan zebrafish optik şeffaflık, 3β-Hsd histokimya başarıyla sabit Zebra balığı embriyo ve larva için kullanılabilir tüm montaj ve özellikle farklılaşmış interrenal dokuları tasvir. Bu, hassas ve hızlı bir deney interrenal dismorfogenezis farklı gösteren Çeşitli mutantlar ve morphants uygulanmıştır. Interrenal 3β-HSD &#39;aktivitesi interrenal doku dağılımı Ff1b transkripsiyon faktörünün belirli bir devirme yoluyla bozulur ve interrenal farklılaşma Ff1b coregulator Prox1 9,10 bir devirme etkilenir azaltılır embriyoda yoktur. Özellikle, 3β-Hsd faaliyeti gibi tek gözlü toplu iğne başı, şiddetli erken evre defekti olan mutantlar tespit edilebilir ve nerede 3β-H, şaşılıksd histokimyası interrenal hücre göçü 11 nasıl etkilendiğini anlatır. interrenal doku farklılaşması bile kan ve damarsal tam yokluğunda ödün verilmemiş. Bu nedenle endotelyum türevli sinyalleri şekil kadar geliştirilmesi interrenal organı 12,13 belirlenebilir. Genel olarak, bu histokimyasal deney başarıyla zebrabalıkları modelinde özellikleri, farklılaşmasını ve steroidojenik hücrelerinin göçünü incelemek için kullanılmıştır. Bu nedenle, verimli ve adrenal ve interrenal organ bozuklukları hedefleyen herhangi bir genetik veya kimyasal ekranlar için güvenilir bir aracı olmalıdır.

<table><tbody><tr><td>Confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td>LSM510&amp;nbsp;</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>USB</td><td>US16374</td><td></td></tr><tr><td>Hyclone Fetal Calf Serum&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30073</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma</td><td>N1636</td><td></td></tr><tr><td>4-Nitro blue tetrazolium</td><td>Promega</td><td>S380C</td><td></td></tr><tr><td>Nusieve GTG</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phenylthiourea</td><td>Sigma&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-100Tab</td><td></td></tr><tr><td>PYREX Spot Plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Reef Salt</td><td>AZOO</td><td>AZ28001</td><td></td></tr><tr><td>trans-Dehydroandrosterone</td><td>Sigma</td><td>D4000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000M</td><td></td></tr></tbody></table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

Pronephric böbrek glomerül ve doğmakta olan damarsal ile steroidogenik interrenal doku codevelops, 3β-Hsd enzimatik aktivite deneyi çift transgenik Tg (wt1b: GFP) üzerinde gerçekleştirildi nasıl belirlemek için LI1; Tg (kdrl: mCherry) ci5 embriyo 34 hpf (Şekil 1). Bu aşamada, 3β-Hsd aktivitesi pozitif steroidogenik doku sağ orta hatta yer alan ve bazı steroidogenik hücreler orta hatta genelinde göç başlar ve dors...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Zebra balığı interrenal bezi memeli adrenal bezin teleostean karşılığıdır. Enzimatik aktivite deneyi, gelişmekte olan Zebra balığı farklılaşmış steroidogenik hücreleri tespit; Bu protokol 3-β hidroksisteroid dehidrojenaz (3β-Hsd Δ 5-4 izomeraz) nasıl gerçekleştirileceği tanıtır.

Interrenal steroidogenik Doku ve Zebra balığı Onun Vasküler Mikroçevre görselleştirme

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Research

JoVE Journal

Developmental Biology

6.9K Görüntüleme.  Tunghai University.  Zebra balığı interrenal bezi memeli adrenal bezin teleostean karşılığıdır. Enzimatik aktivite deneyi, gelişmekte olan Zebra balığı farklılaşmış steroidogenik hücreleri tespit; Bu protokol 3-β hidroksisteroid dehidrojenaz (3β-Hsd Δ 5-4 izomeraz) nasıl gerçekleştirileceği tanıtır. 

Video: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

diseksiyon mikroskobu kullanılarak Time-lapse Görüntüleme ile Zebra balığı Böbrek Kalkınma Analizi Optik Kesit için donatılmış

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Gelişim Biyolojisi

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

Tüm Dağı Zebra balığı Embriyolar kirpikli Organları boyanmasına Fosfo-epitoplar

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Kimya

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Larva Zebra balığı Gonadal Doku Diseksiyon

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Zebrabalığı Tümör Ksenogreftlerinde Konak Anjiyojenik Yanıtın Vivo Görüntüleme ve Kantitasyonunda

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Kanser Araştırmaları

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Zebra balığı bütün Dağı Floresan Situ hibridizasyon ve ayirt kombine bir protokol aracılığıyla Multiciliated hücrelerde görüntülenmesi

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Kemik doku Yenileyici prednizolon etkilerini incelemek için yetişkin zebra balığı yaralanma modelleri

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

Brightfield Mikroskobu Kullanan Larva Zebrabalığında Karaciğer Boyutunun Ölçülmesi

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

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 ...

Biyoloji

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diyabet ile gözleri bir balık eğitim: mikrodiseksiyon, görselleştirme ve yetişkin tg(fli:EGFP) Zebra balığı retina damarlara analizi

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Tıp

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Tüm Dağı ile Zebra balığı Embriyo örnekler Vitray gözlenmesi ve Analiz Daire Dağı Hazırlık<em&gt; In situ</em&gt; Melezleme

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...