Bu yöntemler, JT, laboratuar Destek DePaul Üniversitesi Araştırma Konseyi ve JT (DE016678) NIH / NIDCR R01 hibe tarafından sağlanan bir araştırma izni sırasında EEL tarafından geliştirilmiştir. Biz minnetle Caroline Hunter, CMRC zebrafish çekirdek tesiste hayvan bakım teknisyeni lisans laboratuvar asistanı ve Paulina Pawluczuk çabaları kabul etmiş sayılırsınız.

Zebra balığı yetiştiriciliği Zebra balığı, 1 standart yöntemler göre ev sahipliği ve yetiştirilir . Geçmiş geç larva döneminde, bir zebrafish gelişimsel aşaması, kuyruk yüzgeci (posteriormost hypural plaka) tabanı standart uzunluğu üst dudak anteriormost noktası (SL) veya milimetre olarak düz bir çizgi mesafe ölçülebilir 2. Yaklaşık bir ay sonrası fertilizasyon (10-12 mm SL), burun ve maksiller barbels sırasıyla, koku çukurların yanında ve arka köşelerinde radyografları epitelyal tomurcuklar gibi şeffaf görünür. Zebra balığı olgunlaştıkça, barbels dar, bıyık gibi uzantıları içine uzanır. Maksiller Bärbel büyük (2-3 mm) ve işlemek için kolay olduğundan, protokolü sadece bu özel apendiks için geçerlidir; benzer teknikler, ancak, küçük burun Bärbel adapte olabilir. Bärbel Kırpma <ol><li> Sistem suyun pH 7.0 tamponlu (etil 3 aminobenzoate methanesulfonate) batırılarak istenilen aşamada (örneğin, 1.5-2.5 cm SL, ~ 3-6 ay sonrası fertilizasyon)% 0.015 MS-222 yetişkin Zebra balığı uyuşturan. Durdurana kadar yüzmeye hareketleri gözlemleyin ve solungaç havalandırma yavaş ve düzenli bir hale gelir. Balık büyüklüğüne bağlı olarak, tam bir anestezi yaklaşık 2-5 dakika sürer. </li><li> Bir akvaryum balık ağı kullanarak, bir defada 1-3 balık transferi bir Petri kabındaki ıslak kağıt havlu katlanmış bir parçası. Künt bir spatula kullanarak, şark onlar kadar birbirlerine ve sol lateral yan paralel şekilde balık. Katlama, işlem sırasında cilt ve solungaçları nemli tutmak için operculum&#39;un kadar tüm vücudu kaplayan balık, (solungaç kapakları) kaudal parçası üzerinde ıslak bir kağıt havlu parçasıdır. </li><li> Kafası bölgeyi aydınlatmak için düşük açılı ışık kullanarak, stereomicroscope altında Petri plaka. Maksillanın arka ventral köşesi (Şekil 1) çıkıntı sol maksiller Bärbel kaide üzerinde odaklanın. <img src="/files/ftp_upload/1558/1558fig1.jpg" alt="Şekil 1" /> Şekil 1. </li><li> Sıkıca Bärbel maksillanın kenarına hafifçe distal şaft kavramak. Bärbel mili Elevate ve forseps sadece proksimal ince uçlu bir yaylı makas çeneleri eklemek. Makas istikrar için forseps dokundu olabilir. Kesme kenarı maksilla kenarına yaklaşana kadar Bärbel mil boyunca makas çeneleri kaydırın. Bärbel mili kesmek için makas kapatın. Kesilmiş karakeçi, hala forseps kavradı sabitleme ya da daha fazla gözlem için çıkarılabilir. </li><li> Hemen bir damla metilen mavisi yüzeysel mantar enfeksiyonu kontrol etmek için içeren temiz su (~ 500 ml) küçük bir tank için balık transferi. Balık bir gecede kurtarmak için izin ver. Ertesi sabah, balık yetiştirme sistemi dönün. Bärbel rejenerasyon 2 hafta sonra gözlenir. </li></ol> Uyumlu Bärbel Çiftlerin Agar gömülmesi <ol><li> Uygun ötenazi tekniği ile balık toplayın ve ajitasyon ile 4 ° C gecede tamponlu salin (PBS)% 4, 50 ml tüp paraformaldehid-fosfat düzeltmek. Fiksatif hacmi en az on kez doku hacmi olmalıdır. Fiksasyon sonra, onaylanmış bir kapta paraformaldehid atık imha ve doku PBS çeşitli değişiklikler iyice durulayın. </li><li> % 2 250 ml cam şişesi 150 ml hazırlayın agaroz (T m ~ 37 ° C) distile su. Tamamen eriyene kadar bir mikrodalga veya sıcak plaka ile Isı, periyodik olarak ajitasyon. 50-60 ° C su banyosu içinde olan erimiş çözüm dengelenmesi. </li><li> Standart bir jel elektroforezi teçhizat sıkıca tampon odasının yanında karşı küçük bir contalı jel kalıp (~ 100 ml jel hacmi 10 x 10 cm) koyarak birleştirin. 2-4 küçük dişli örnek tarak (4 x 1.5 mm). </li><li> Bir yüzey üzerinde, çok sığ kuyular oluşturan, tarak tabanı kapsayacak sadece kalıp içine erimiş agaroz dökün. Hemen geri kalan agaroz sıcak su banyosu içine koymak, bu doku (9-12 Adımlar) gömmek için daha sonra kullanılabilir olacak. Pipet agaroz aynı sıcaklığı tutmak için uzun bir cam Pasteur pipeti balonun içine koyun. </li><li> 20-30 dakika sertleşmesi için jel izin verin. Tarak çıkarın, sonra tampon odasından sertleştirilmiş jel içeren jel kalıp çıkarmak. Jel dışarı slayt değildir laboratuvar bant ile jel kalıbın açık tarafı sıkıca bantlayın. Bu bant ek agaroz (Adım 13) daha sonra eklenebilir böylece agaroz yüzey üzerinde birkaç milimetre uzatmak gerekir. </li><li> Bir stereomikroskopta aşamasında jel kalıp yerleştirin. Boş bir iyi büyütmenize ve kenarları odak noktası haline getirmek. </li><li> Iyi komşu agaroz yüzeyine bir damla su veya sabit Bärbel örnekleri (örneğin, yeniden ve kontrol) içeren tampon pipetle. USIng nemli örneklerdir kuyunun içine sürükleyin ve onları aşağıya doğru itin, böcek iğne eğildi. Orient Bärbel milleri birbirine paralel ve iyi zıt uzun kenarlarına dizilen. Yüzey gerilimi, doku pozisyonda tutun yardımcı olacaktır. </li><li> Gömmek için hazır olduğunuzda, ince bir pipet veya aşırı sıvı kaldırmak için iyi bir laboratuvar doku köşe kullanın. Doku kurumasına izin vermeyin hızla çalışın. </li><li> Isıtılmış cam Pasteur pipeti kullanarak, yavaşça yerine mühür Bärbel doku örneklerinde ve çevresinde taze erimiş agaroz pipetle. Doldurmayın. Agaroz sertleşir önce, böcek iğneli son dakika ayarlamaları yapmak. </li><li> Iyi yanında numaralı bir kağıt numune etiket yerleştirin ve agaroz küçük bir damla ile sabitleyin. </li><li> Numunelerin her bir kuyu için 6 - 10 arasındaki adımları tekrarlayın. </li><li> Görüntü kalibrasyonu için İsterseniz, baskılı mikrometre ölçeğinde (örneğin, bir parça kağıt yerleştirin <a href="http://incompetech.com/graphpaper/multiwidth/">http://incompetech.com/graphpaper/multiwidth/</a> de boş bir dibine). Kuyunun dibine kağıt düzleştirin ve sıcak agaroz ile örtün. </li><li> Tüm örneklerin gömülü sonra, agaroz jel yüzey sertleştirilmiş damla, düzensiz olacaktır. Düz bir yüzey üzerinde jel yerleştirin ve üst yüzeyi tekdüze ek erimiş agaroz kadar yavaşça dökün. Düzgün bir yüzey elde etmek için gerekli agaroz en az miktarda kullanın; çok agaroz iletilen ışık fotoğrafçılığı belirsiz. Bu üst tabaka sertleşmesine izin verin. </li><li> Jel şimdi her Bärbel çifti için brüt morfolojisi kaydetmek için bir stereomikroskopta aşamasında fotoğrafı olabilir. Resim kalibrasyonu gömülü mikrometre ölçeğinde fotoğrafını elde edilir. </li><li> Tüm jel, ıslak kağıt havlu sarılır ve birkaç hafta boyunca 4 ° plastik bir torba içinde mühürlü saklanabilir. </li><li> Ayrıntılı analiz için jel agar blokları barbels eşlemeli çifti içeren bir neşter veya jiletle kesip olabilir ve parafin histoloji işlenen veya standart yöntemler ile 3,4 cryosectioning. Alternatif olarak, bireysel barbels suda durulanır ince iğneler, agaroz disseke ve diğer downstream uygulamalar (örneğin, tüm montaj immünohistokimya veya konfokal mikroskopi) işlenmiş olabilir. </li></ol>

<ol>
	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1+General+Methods+for+Zebrafish+Care.">Chapter 1 General Methods for Zebrafish Care.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Howe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+length:+Not+quite+so+standard.">Standard length: Not quite so standard.</a> Fisheries Research (Amsterdam). 56, 1-7 (2002).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+Tissue+Samples+for+Sectioning.">Paraffin Embedding Tissue Samples for Sectioning.</a> Cold Spring Harbor Protocols. 6, (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+8.3+Agar+Embedding+for+Cryostat+Sectioning+of+Embryos+or+Larvae.">Ch 8.3 Agar Embedding for Cryostat Sectioning of Embryos or Larvae.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Hansen, A., Reutter, K., Zeiske, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+bud+development+in+the+zebrafish,+Danio+rerio.">Taste bud development in the zebrafish, Danio rerio.</a> Dev Dyn. 223, 483-496 (2002).</li></ol>

Zebrafish maksiller Bärbel zebrafish çeşitli hücre tiplerinin büyüme, bakım ve yenilenme eğitim için atıl doku sistemi. Bärbel apendiks hiçbir insan analog olmasına rağmen, içerdiği hücre tipleri çok mümkün optik açık ve anatomik basit bir silindirik yapıdaki cilt bezleri, melanosit, dolaşım damarları ve sinirleri çalışmak için korunmuş bulunmaktadır. Iyi çalışılmış bir kuyruk yüzgeci, Bärbel doku amputasyon tarafından yeniden başlatılabilir. Maksilla sınır anatomik bir dönüm noktası olarak kullanarak, amputasyon düzlemde Bärbel büyütme ölçümü kolaylaştırmak, hassas şekilde yerleştirilmiş olabilir. Deneyimli bir operatör için, her ameliyat, sadece birkaç saniye sürer. Kurtarma hızlı ve balık davranışları üzerinde herhangi bir kısa ya da uzun vadeli etkileri şu ana kadar tespit ettik. Zebra balığı, bir maksiller Bärbel yüzerek cerrahi sonrası 6 aya kadar, yemek ve cerrahi olmayan kontroller olarak etkin bir şekilde doğurmak ve doku toplama süresi ile karşılaştırılabilir bir yaşam süresine sahiptir. Bu ameliyatı fizyolojik etkileri, 1) Bärbel yürütülen ekstraoral tat tomurcukları da balık epitel dudaklar, yanaklar ve yüksekliği 5 de dahil olmak üzere diğer birçok parça bulunur, çünkü çok az olacağı tahmin, ve 2) ayırt tat ​​tomurcuklarının görünür 72 saat içinde yenileyici Bärbel (Leclair ve ark. yayınlanmamış veriler). maksiller Bärbel yara iyileşmesi, revaskülarizasyon ve reinnervation bir yetişkin omurgalı kapsamında eğitim için minimal invaziv bir sistem yapar. Rejenerasyon cerrahi indüksiyonundan sonra, sabit doku morfometrik ölçüm ve / veya mikroskopik analiz için barbels aralıklarla toplanmalıdır. Elverişli, maksiller Bärbel cryosectioning, tüm montaj immünhistokimya ve in situ, parafin histoloji dahil olmak üzere birçok standart protokoller, uygulanmasını kolaylaştırmak için, yaklaşık uzunluğu (2-3 mm) ve bir zebrafish embriyonun çapı (100-200 mm ). melezleşme. Birlikte ele alındığında, bu özellikleri maksiller Bärbel in vivo model, doku onarımı ve yenilenmesi eğitim için son derece gerçekçi olun .

Mikroskop Ekipman Aktarılır ve olay fiber optik aydınlatma ile stereo diseksiyon mikroskobu Bärbel kırpma ve agar gömme için gerekli. Bärbel Kırpma <ul><li> Balık sistem su </li><li> 2 zebrafish geçiş tankları </li><li> MS-222 (etil 3-aminobenzoate methanesulfonate; Sigma Chemical # E10521-10G) </li><li> 120 mm steril Petri plaka </li><li> ıslak kağıt havlu </li><li> künt metal spatula </li><li> Dumont # 55 Bio Inox Forseps (Güzel Bilim Araçları # 11.255-20) </li><li> Mini-Vannas yaylı makas (Güzel Bilim Araçları # 15.000-00) </li><li> metilen mavisi antifungal ajan (Drs. Foster &amp; Smith # CD-905.781) </li><li> küçük akvaryum fishnet </li></ul> Doku toplama ve agar gömme <ul><li> 1x 4% paraformaldehid fosfat tamponlu salin (PF-PBS), 50 ml konik tüpler içine aliquoted ve -20 ° C arasında dondurulup saklanan kadar kullanımda </li><li> 1x fosfat tamponlu salin (pH 7,27-6) </li><li> 250 ml cam şişesi </li><li> % 2 agaroz distile su içinde </li><li> standart küçük agaroz jel elektroforezi teçhizat (jel boyut = ~ 10 cm kare) </li><li> küçük dişli elektroforez örnek tarak (tarak boyutu 4 x 1,5 mm) </li><li> sıcak su banyosunda (50-60 ° C) </li><li> laboratuvar bant </li><li> siyah böcek iğne, boyut 000 (Güzel Bilim Araçları # 26.000-25) emaye </li><li> 1-2 pin sahipleri (İnce Bilim araçları # 26.016-12) </li><li> 9-inç cam Pasteur pipeti </li><li> pipet ampul veya pompa </li><li> laboratuvar doku </li><li> küçük kağıt etiketler </li><li> (Isteğe bağlı) basılı kalibrasyon ölçekli </li><li> ıslak kağıt havlu </li><li> zip kapanması plastik torba </li><li> neşter / jiletle (agar blokları kesip) </li></ul>

methods for the study of the zebrafish maxillary barbel

Barbels cilt duyusal uzantıları, balıklar, sürüngenler ve amfibiler bulundu. Zebra balığı Danio rerio barbels-kısa bir burun çifti ve uzun maksiller çiftinin iki çift geliştirir. Bärbel doku, deri hücreleri, salgı bezleri, tat tomurcukları, melanositler, dolaşım damarları ve duyusal sinirler dahil olmak üzere ektodermal, mezodermal ve nöral krest kökenli hücreler içerir. Çoğu yetişkin doku aksine, maksiller Bärbel, bize yaşam döngüsü boyunca bu doku tiplerinin geliştirme ve bakım görselleştirmek için izin, optik açıktır. Bu video maksiller Bärbel (yaklaşık bir ay sonrası döllenme başlamadan) erken gelişim gösterir ve yenilenme yetişkin apendiks (&gt; 3 ay sonrası fertilizasyon) ikna etmek için bir cerrahi protokol göstermektedir. Kısaca, bir anestezi balık sol maksiller karakeçi maksilla kaudal kenarı hemen distalinde steril forseps ile yükselir. Ince, steril bir yaylı makas amputasyon düzlemde anatomik için bir dönüm noktası oluşturan bu seviyede Bärbel mili kesmek için forseps karşı konumlandırılmış. Rejeneratif büyüme ve kontralateral Bärbel karşılaştırıldığında bu uçağa göre ölçülebilir. Bärbel doku yaralanma 2 hafta içinde maksimum regrowth ulaşan hızla yeniler. Rejenere Bärbel analiz teknikleri, standart bir DNA elektroforez jel kuyulardan barbels eşli çiftler (yeniden ve kontrol) diseksiyon ve gömme içerir. Gömülü örneklerin uygun bir brüt morfolojisi ve morfometri stereomikroskop altında fotoğraflandı ve parafin histoloji gibi downstream uygulamaları öncesinde hafta boyunca saklanan cryosectioning ve / veya tüm montaj immünohistokimya olabilir. Bu yöntemler, in vivo doku sistemi, birden fazla hücre tiplerinin yenileyici kapasitesi zebrafish genetik kapsamında eğitim için bir roman olarak maksiller karakeçi kurmak .

Watch this Scientific Journal Video about Methods for the Study of the Zebrafish Maxillary Barbel at JoVE.com

Methods for the Study of the Zebrafish Maxillary Barbel

Zebrafish maksiller Bärbel ektodermal, mezodermal ve nöral krest türevleri içeren bir kabukla duyu organ. Önemlisi, yetişkin Bärbel proksimal amputasyon sonra yeniden oluşturabilirsiniz. Bu video maksiller Bärbel gelişimi tanıtır ve Bärbel örneklerin toplanması, gömme ve alt görüntüleme ile takip rejenerasyon ikna etmek için bir cerrahi protokolü göstermektedir.

Zebra balığı Maksiller Bärbel Çalışma Yöntemleri

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

methods-for-the-study-of-the-zebrafish-maxillary-barbel

Research

JoVE Journal

Biology

12.4K Görüntüleme.  DePaul University. Zebrafish maksiller Bärbel ektodermal, mezodermal ve nöral krest türevleri içeren bir kabukla duyu organ. Önemlisi, yetişkin Bärbel proksimal amputasyon sonra yeniden oluşturabilirsiniz. Bu video maksiller Bärbel gelişimi tanıtır ve Bärbel örneklerin toplanması, gömme ve alt görüntüleme ile takip rejenerasyon ikna etmek için bir cerrahi protokolü göstermektedir.

Video: Methods for the Study of the Zebrafish Maxillary Barbel

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biyoloji

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Bilgisayar oluşturuldu Hayvan Modeli Uyaranlar

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Zebrafish görme fonksiyonu çalışma optokinetik yanıt

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Yetişkin Zebra balığı içine intraperitoneal enjeksiyon

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Zebra balığı Pronephros Lazer Ablasyon Böbrek Epitel Rejenerasyon Eğitim

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

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 ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

KRİYOPREZERVE Numune Kurtarma için Do-It-Yourself Aygıt yanlışlıkla Kriyojenik Depolama Tankları içine Dropped

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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 ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Beyaz sinekten Endosimbonların Çıkarılması İçin Yöntemler<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...