Name: immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos
Uploaded: 2016-02-19T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Bu çalışma, Ulusal Bilim Vakfı hibe IOS-0817658 tarafından desteklenmiştir.

Bu protokol zebrabalıkları prosedürler Virginia Commonwealth Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. Reaktiflerin 1. Hazırlık <ol><li> % 4 PFA / PBS ile yıkandı. davlumbaz paraformaldehit 8 g (PFA) tartılır. Hala davlumbaz, ~ 80 ml ​​50 ° C, karıştırma ve ısıtma ile damıtılmış H2O kuru PFA çözülür ise. PFA tamamen çözülene ve çözelti açıklık kadar taze 1 N NaOH 10 damla - karıştırılırken, 3 ekleyin. Ateşten ve 100 ml 2x fosfat tamponlu tuz (PBS) ilave edin. Damıtılmış H2O 200 ml hacim getirmek Oda sıcaklığına soğutun ve kullanımdan önce 7.4 pH teyit etmektedir. 4 ° C de karanlıkta deposu ancak bir hafta içinde kullanın. </li><li> herhangi bir ek olmadan% 100 metanol kullanın. herhangi bir ek olmadan% 95 etanol kullanın. </li><li> Fosfat tamponlu Tween (PBT) hazırlayın. % 0.1 Tween-20 ile fosfat tamponlu tuz (PBS) Supplement. 0 ekleyin.PBS 1X 100 ml% 20 Tween-20 stok çözeltisi 5 mi. Oda sıcaklığında saklayın. </li><li> Fosfat tamponlu Triton-X (PBTx) hazırlayın. % 0.1 Triton X-100 ile PBS Supplement. PBS, 100 ml bir% 20 Triton X-100 stokunun 0.5 ml ilave edilir. Oda sıcaklığında saklayın. </li><li> % 10 NGS / PBTx hazırlayın. Normal keçi serumu (NGS) blok spesifik olmayan bağlanma siteleri. -20 ° C&#39;de parçalar halinde saklayın NGS. Kullanmak 9 ml PBTx 1 ml ekleyin. </li><li> % 50 gliserol / PBS hazırlayın. 2x PBS ve% 100 gliserol iyice eşit parçaya karıştırın. Oda sıcaklığında saklayın. </li></ol> 2. Embriyo Sabitleme <ol><li> . (., Örneğin β-aktin: CAAX-GFP) Damızlık elde vahşi tip (AB ve WIK) veya transgenik doğal çiftleşmelerin aracılığıyla embriyolar. İstenirse açıklandığı gibi, yapıları veya morpholinos ile embriyolar enjekte edilir. 15,16 </li><li> . Embriyolar kaldırın embriyolar toplayın ve tarif edildiği gibi pigmentasyon engellemek için% 0.003 1-fenil-2-tioüre (PTU) mevcudiyetinde 28.5 ° C&#39;de inkübe edin. 17 </su<html> p&gt; </li><li> Embriyolar istenilen gelişim aşamasına, 18 ulaştığında. Fix, &#160; , MESAB kullanılarak embriyolar anestezi mümkün olduğu kadar sistem, suyu buharlaştırmak ve 3 taze% 4 PFA / PBS ekleyin - oda sıcaklığında 4 saat. NOT: 20 saat sonra döllenme (hpf) daha az zamanlarda, dechorionation önce düzeltin. 20 hpf sonra zaman zaman, bir mikroskop altında ince nokta forseps iki çift kullanarak fiksasyon önce dechorionate. </li><li> Çözümler değiştirme. Geniş bir delik pipet kullanarak embriyoların transferi, tarif edildiği gibi. Mikrosantrifüj tüpleri şapkalı 1.5 ml 17 Değişim çözümleri, sadece embriyolar santrifüj olmadan yerçekimi ile yerleşmek için izin vererek. </li><li> Sonrası sabitleyici sonra 3 -. 4 saat, en az 48 saat boyunca -20 ° C&#39;de% 100 metanol ve mağaza ile yerine, PFA / PBS kaldırmak. NOT: maksimal P-CAMK-II immünoreaktivitesinde için, bir hafta toplam metanol saklama süresini sınırlamak. </li></ol>&quot;&gt; 3. Immün Tüm Embriyolar <ol><li> başlıklı bir 1.5 ml mikrosantrifüj tüpü içinde, her deneysel koşul için 10 embriyo az yerleştirin ve etiketlemek. </li><li> embriyolar yerleşmek için izin verin. Çıkarın ve metanol atmak ve bir sonraki aşamada gösterildiği gibi, etanol ve azalan konsantrasyonlarda 0.5 ml ihtiva eden aşamalı yıkama ile rehidrate. Her adım sallanan bir 5 dk yıkama olduğunu. </li><li> Kademeli bu 5 çözümleri ile rehydrate:% 66 etanol /% 33 PBTx,% 33 etanol / PBTx,% 100 PBTx,% 100 PBTx (bu PBTx ilk tekrarıdır),% 100 PBTx (bu ikinci tekrarı ise% 66 PBTx). </li><li> Geçen PBTx yıkama çıkarın. 0.5 ml% 10 NGS / PBTx ekleyin. hafifçe çalkalanarak oda sıcaklığında en az 1 saat boyunca inkübe edilir ve akabinde çıkarın ve solüsyonu atın. </li><li> % 10 NGS / PBTx içinde seyreltilmesi birincil antikor çözüm hazırlayın. CO-immün, önce yüksek afinite antikor kullanımı. 500: Bu çalışma için, 1 seyreltilmiş fare anti-asetile tübülin monoklonal antikor ile inkübe edin. exampl içinE, 10 numune,% 10 NGS / PBTx 3 ml stok antikor çözeltisi 6 ul birleştirilir ve daha sonra, her tüpe, bu, 0.3 ml dağıtmak varsa. </li><li> Tüm embriyo sokmak için her bir tüpe seyreltilmiş birincil antikor 0.5 ml - 0.2 ekleyin. Oda sıcaklığında nazik sallanan O / N ile kuluçkaya yatmaktadır. </li><li> Sabah, kaldırmak ve antikor çözüm atın. Fazla birincil antikor yıkayacak 0.5 ml% 2 NGS / PBTx ekleyin. Yavaşça 5 dakika sallayın. Çıkarın ve çözüm atın. İki kez tekrarlayın. </li><li> tepe ışıkları Dim ve her bir durum, en azından 0.5 ml içerir, böylece% 10 NGS / PBTx uygun floresan-konjuge sekonder antikor seyreltin. : 500 oranında seyreltilmiş fare anti-asetillenmiş tübülin birincil antikor ile, 1 yeşil fluoresan boya keçi anti-fare IgG kullanın. </li><li> yumuşak karanlıkta oda sıcaklığında çalkalanarak 4 saat sekonder antikoru inkübe edin. silik ışıkları altında ve karanlık bir kapta inkübe veya alüminyum folyo ile sararak numuneler süreç, şu andan itibaren. </li><li>İnkübasyonun sonunda, kaldırma ve ikincil antikor solüsyonu atın. 0.5 ml% 2 NGS / PBTx yıkamak ekleyin. Yavaşça 5 dakika sallayın. Çıkarın ve çözüm atın. İki kez tekrarlayın. </li><li> CO-immün, önce birincil antikor daha farklı bir türden gelen ikinci birincil antikor elde edilir. Bu çalışmalarda, kırmızı fluoresan boya-konjüge edilmiş keçi anti-tavşan IgG ikincil antikor tavşan anti-fosfo CAMK-II antikoru izleyin. </li><li> tavşan anti-fosfo CAMK-II antikoru 01:20 sulandırmak. Embriyoların her bir tüp için, daha sonra, 0.3 ml% 10 NGS / PBTx askıya hazır antikor çözeltisi 15 ul ekle. </li><li> Embriyolar dalmış ve ışıkları sönük olduğundan emin olun. karanlıkta oda sıcaklığında hafif sallanan O / N ile kuluçkaya yatmaktadır. </li><li> loş ışıklar altında Sabah, kaldırmak ve antikor çözüm atın. 0.5 ml% 2 NGS / PBTx ekleyin. Yavaşça 5 dakika sallayın. Çıkarın ve çözüm atın. İki kez tekrarlayın. </li><li> loş havai ışıkları tutmak ve uygun floresan konjugat yeterince sulandırmakkapı ikincil antikor, böylece her koşul en az 0.5 ml içerir. 500,% 10 NGS / PBTx: Bu çalışmada, kırmızı fluoresan boya-konjüge edilmiş keçi anti-tavşan IgG ikincil antikor (kırmızı kanal) 1 seyreltin. </li><li> yumuşak karanlıkta oda sıcaklığında çalkalanarak 4 saat, ikinci ikincil antikor inkübe edin. </li><li> Bu inkübasyon ve loş ışıkları altında sonunda, kaldırmak ve ikincil antikor çözüm atın. 0.5 mi PBTx ekleyin. Yavaşça 5 dakika sallayın. Çıkarın ve çözüm atın. İki kez tekrarlayın. Mağaza PBTx ya da% 50 gliserol / PBS ya görüntüleme işlemi bağlı olarak değişir. </li></ol> 4. Konfokal Görüntüleme ve İşleme <ol><li> Dağı görüntüleme için embriyolar. Bir bardak slayt 5 embriyo - 1 yerleştirin. Ana lamel ve odanın her iki tarafında lamel fragmanları kullanılarak kızak arasında bir bölme oluşturur. Tipik olarak, dört 1. lamelleri sızdırmazlık olmadan bu boşluk yığınları yapmak için kullanılır. NOT: Hiçbir montaj orta gereklidir. </li><li> Bir 100X yağ daldırma kullanınAmaç iletilen ışık kullanarak odak noktası haline tek embriyo getirmek. Işığı kapatın. konfokal mikroskop açın. Bu uygun lazerler (yeşil ve / veya kırmızı) açık ve uzaktan odak accessary oturduğundan emin olun. </li><li> konfokal programı açın. In &quot;Edinme bar,&quot; Doğru hedefi seçin. &quot;XY Temel&quot; çubuğunda, 1.024 butonuna tıklayarak 1.024 görüntü boyutunu ayarlayın. </li><li> &quot;Lazer ve Dedektörü&quot; barda, her kanal için lazer ve dedektör açmak için kırmızı 488 kutu ve yeşil 568 kutusuna tıklayın. orta iğne deliği ayarlayın ve görselleştirmek için her kanalın &quot;Kazanç&quot; barda kazancını ayarlayın. </li><li> &quot;Elde Ayarlar&quot; çubuğunda, görüntüleri elde başlamak için &quot;Canlı&quot; seçeneğine tıklayın. &quot;Görünüm Ayarları&quot; çubuğunda, &quot;Canlı&quot; penceresinde merkezine &quot;İntegral Yakınlaştırma zorla&quot; kutusunun işaretini kaldırın. </li><li> &quot;Ayarlar Edinme&quot; barda &quot;Z&quot; sekmesi altında, görüntünün katmanları üzerinden adım. nu seçtikten sonraOptik bölümden mber, görüntü içine dahil z-düzlemi &quot;Merkezi&quot; bazı noktaya taşımak için. </li><li> &quot;Z&quot; sekmesi altında, küçük kırmızı &quot;Referans&quot; kutusuna tıklayın. Bu &quot;merkez&quot; olarak seçilen noktada RFA sıfır olacaktır. &quot;Adım Boyutu&quot; etiketli kutuda (ideal mikron mikron 0.25 ila 2.0) katmanların kalınlığı seçin. &quot;Dosya Boyutu&quot; kutusuna dikkat edin ve 1GB sınırlamak için deneyin. Not: Genellikle, 0.5, 40, optik bölüme kadar - 1.0 um elde edilir, ancak bölme sayısı deneysel olarak tespit edilebilir. </li><li> Resmin üst extreme bulmak için, &quot;Top&quot; yanındaki daireyi tıklayın ve z katmanları arasında hareket. Şimdi, bir sonraki &quot;Bottom&quot; için daireyi tıklayın bilgisayar merkezi olarak katmana geri görüntü alacaktır. Yine resmin alt aşırı bulmak için katmanları arasında hareket. </li><li> lazer durdurmak için kutu &quot;Edinme Ayarlar&quot; tekrar &quot;Canlı&quot; linkine tıklayınedinimi. Bu panelde, kırmızı Ortalama etiketli kutuları ve Z-yığını tıklayın. Bu kutular yeşile döner. </li><li> &quot;Elde Ayarlar&quot; kutusunda, görüntülerin tüm seriyi kazanmak için &quot;Single&quot; tıklayın. her iki kanalda ve tüm z-yığın içinde tarar gibi ilerlemesini izleyebilirsiniz. </li><li> Kaydetmek birimi penceresinde tıklayın ve tıklayın &quot;farklı kaydet&quot; ve dosyayı adlandırın. Bir &quot;.ids&quot; dosyası olarak kaydedin. hacmine z yığın açıkken, dosyayı işlemek Veri menüyü aşağı çekin ve &quot;render hacim&quot; linkine tıklayarak seçin. Bir tiff dosyası olarak verilen dosyayı kaydedin. Bu, ilgili yayının gösterilmiştir yansıtılan görüntüdür. </li></ol>

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Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

PFA / metanol yöntemi zebrabalıkları gelişim sırasında fosfo-T 287 -CaMK-II epitopunun bağışık optimize temel amacı ile bu laboratuvarda geliştirilmiştir. Bu yöntem başarıyla zebrabalıkları KV, 14 ve özellikle KV aşamada böbrek. 13, bu tekniğin gerekli olduğu 12 iç kulak olmak üzere birçok siliyalı organların oluşumu sırasında P-CAMK-II lokalize. Bu yöntemin başarısı otofloresansı a) en aza indirilmesi, fosfo-CAMK II epitopunun b) korunması...

The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals. 
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred. 
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer's Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer's Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.

<table><tbody><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma</td><td>P-7629</td><td>0.12% Stock solution. Dilute 1:40 in system water</td></tr><tr><td>Alexa488 anti-mouse IgG</td><td>Life Technologies</td><td>A11001</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 anti-rabbit IgG</td><td>Life Technologies</td><td>A11008</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 phalloidin</td><td>Life Technologies</td><td>A12379</td><td>Preferentially binds to F-actin</td></tr><tr><td>Alexa568 anti-mouse IgG</td><td>Life Technologies</td><td>A11004</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa568 anti-rabbit IgG</td><td>Life Technologies</td><td>A11011</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>anti-acetylated &amp;#945;-tubulin</td><td>Sigma</td><td>T7451</td><td>Mouse monoclonal, use at 1:500</td></tr><tr><td>anti-phospho-T&lt;sup&gt;287&lt;/sup&gt; CaMK-II</td><td>EMD Millipore</td><td>06-881</td><td>Rabbit polyclonal, use at 1:20</td></tr><tr><td>anti-total CaMK-II</td><td>BD Biosciences</td><td>611292</td><td>Mouse monoclonal, use at 1:20</td></tr><tr><td>Ethanol</td><td>Fisher</td><td>S96857</td><td>Lab grade, 95% denatured</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Dumont #5</td></tr><tr><td>Glass coverslips</td><td>VWR</td><td>16004-330</td><td>#1&amp;nbsp; thickness</td></tr><tr><td>Glass microscope slides</td><td>Fisher</td><td>12-550-15</td><td>Standard glass slides</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A411</td><td>Store in freezer</td></tr><tr><td>Microcentrifuge tubes</td><td>VWR</td><td>20170-038</td><td>capped tubes, not sterile</td></tr><tr><td>Normal goat serum</td><td>Life Technologies</td><td>16210-064</td><td>Aliquot 1 ml tubes, store in freezer</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P-6148</td><td>Reagent grade, crystalline</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Quality Biological</td><td>119-069-131</td><td>10x stock solution or made in lab</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>BP-151</td><td>10% solution in water, store at RT</td></tr><tr><td>Tween-20</td><td>Life Technologies</td><td>85113</td><td>10% solution in water, store at RT</td></tr><tr><td>Compound microscope</td><td>Nikon</td><td>E-600</td><td>Mount on vibration-free table</td></tr><tr><td>C1 Plus two-laser scanning confocal</td><td>Nikon</td><td>C1 Plus</td><td>Run by EZ-C1 program, but upgrades use &quot;Elements&quot;</td></tr></tbody></table>

immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos

hücrelerin hızlı çoğalması, gen dokuya özgü sentezleme ve sinyal ağlar ortaya tüm omurgalıların, erken dönemde embriyo gelişimi karakterize eder. hatta tek hücrelerin içinde - - kinetik ve sinyallerin yeri gelişmekte olan embriyonun önemli gelişimsel genlerin belirlenmesi tamamlar. Immün teknikleri primer kirpikler kadar küçük yapılarda, hücre içi ve bütün hayvan sinyallerinin kinetiklerini belirlemek için gösterilmiştir ki açıklanmaktadır. bir lazer tarama konfokal bileşik mikroskop kullanılarak sabitleme, görüntüleme ve işleme görüntüler için teknikler kadar az 36 olarak saat içinde tamamlanabilir. Zebra balığı (Danio rerio) ekonomik ve insan hastalığı ile ilgili bir omurgalı türlerinde çalışmalar yapmak istiyoruz araştırmacılar için bir arzu organizmadır. Genetik tırnakları veya knockdowns gerçek protein ürününün kaybı ile teyit edilmesi gerekir. protein kaybı Böyle onayBurada tarif edilen teknikler kullanılarak elde edilebilir. sinyal yolları içine ipuçları da çeviri sonrası fosforilasyon ile modifiye edilmiş protein ile reaktif olan antikorlar kullanılarak deşifre edilebilir. Korunması ve bir epitopun fosforile durumunu optimize bu belirleme için kritik öneme sahiptir ve bu protokol ile gerçekleştirilir. Bu çalışma geliştirme ve ilk 72 saat boyunca embriyolar düzeltmek için teknikler anlatılmaktadır Kupffer en veziküllü (KV) &#39;de cilia ile ilgili epitopların çeşitli böbrek ve iç kulak co-lokalize. Bu teknikler diseksiyon gerekmez, basit ve zaman görece kısa bir süre içinde tamamlanabilir. tek bir görüntü içine konfokal görüntü yığınlarının Projelendirme bu verileri sunma yararlı bir araçtır.

Fosfo-epitopları görselleştirme için en uygun koşulları Zebra balığı embriyolarının protein epitoplarının bağışık anlatan Yöntem in situ melezleme yoluyla mRNA&#39;ların lokalize için kıyasla nispeten seyrek olmuştur. Zebra balığı embriyoların silli hücrelerde protein epitoplarının lokalize kullanılan Fiksatifler% 4 PFA / PBS ve metanol ve in situ hibridizasyon b...

Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

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

Teknikler, tüm zebra balığı embriyolar fosfo-epitopları immunostain ve daha sonra birincil kirpikler kadar küçük hücresel yapılarının iki renkli flüoresan konfokal lokalizasyonu yapmak için tarif edilmiştir. tespit ve görüntüleme için teknikler yer ve spesifik protein ortaya veya aktivasyon kinetikleri tanımlayabilir.

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 ...

immunostaining-phospho-epitopes-ciliated-organs-whole-mount-zebrafish

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

8.0K Görüntüleme.  Virginia Commonwealth University.  Teknikler, tüm zebra balığı embriyolar fosfo-epitopları immunostain ve daha sonra birincil kirpikler kadar küçük hücresel yapılarının iki renkli flüoresan konfokal lokalizasyonu yapmak için tarif edilmiştir. tespit ve görüntüleme için teknikler yer ve spesifik protein ortaya veya aktivasyon kinetikleri tanımlayabilir. 

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

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn and 70Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3 and H2O2. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO3 medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique).&#160;This full typical&#160;procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

Fare Organlar Uygulanan Yüksek Hassasiyetli Çinko İzotopik Ölçümler

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

Kimya

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Yüksek Performanslı Sıvı Kromatografi-birleştiğinde Akış Parıldama tarafından Radyoetiketleme ve Fosfoinosititler Hücresel Düzeylerinin Quantification

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

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 ...

Gelişim Biyolojisi

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebra balığı Tüm Dağı Yüksek Çözünürlüklü Çift Floresan Yerinde Hibridizasyon

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

Biyoloji

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Zebrabalığı Embriyoları ve Larvalarında Tüm Mount Immunohistochemistry

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

Zebra balığı Tek Hücre Kader Haritalama

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Zebra balığı Embriyonik Kalp Dissected, immün

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

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Biyokimya

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

In Situ Kriyokesitli Zebra Balığı Embriyolarında İmmünohistokimya ile Kombine Hibridizasyon

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

Nörobilim

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Cryosec, zebrafish embriyo üzerinde sıralı Immünofluorescence ve Immünhistokimya

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...