This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

Bütün hayvan prosedürleri Mie Üniversitesi Etik Kurulu tarafından onaylanmış ve uluslararası kurallara Japon hayvan refahı yönetmelik &#39;Refah ve Hayvanların Yönetimi Yasası&#39; (Japonya Çevre Bakanlığı) göre gerçekleştirilmiştir ve uyulması bulundu. Needle 1. Hazırlık NOT: Bütün deneyler anestezi altında gerçekleştirildi ve tüm çabaları acıyı en aza indirmek için yapılmıştır. Ötenazi için, balık ≥20 dakika boyunca (≤4 ° C&#39;de 5 parça buz / 1 kısım su), bir buz-su banyosu içine daldırıldı. <ol><li> Bir iğne çektirmesi (Şekil 1A) ile 1,0 mm dış çaplı cam kapiller çekerek cam iğneler microcapillary hazırlayın. </li><li> Oblik ince makas (Şekil 1B) kullanarak iğne uçlarını kesin. 200 mikron (Şekil 1C) - İdeal uç çapı yaklaşık 100 olmalıdır. İpucu Diamete Eğerr çok dar, kan iğne girmeyecektir. </li><li> 5 mg / ml &#39;lik bir konsantrasyona kadar tuzla içinde heparin çözülür. </li><li> Bir aspiratör borusu düzeneğinin burunluk sonunda bir precut iğne yerleştirin ve ağız ağızlık tutun, ya da bir ampul dağıtıcı iğne bağlayın. Heparin çözeltisi içine iğne ucu daldırın ve emme ve çözelti (Şekil 1D ve 1E) ile üfleme iğnesi heparinize. Not: aspiratör boru montaj Zebra balığı sperm dondurulması 16 kullanılmıştır. Uzun kauçuk tüp ağzına uçan herhangi bir kan durdurabilirsiniz. Tüpün yarıda bir filtre ayarlama tehlikeleri önleyebilirsiniz. </li><li> En az 1 saat süre ile 10 cm&#39;lik bir Petri kabı ve hava-kuru heparinize iğneler saklayın. Iğneler çok sayıda önceden hazırlanabilir. </li></ol> 2. Anestezi <ol><li> (2-fenoksietanol, 100 ul balık 200 ml su karıştırılarak 2 küçük bir plastik durumda anestezi çözeltisi hazırlayın-PE). Anestezik nihai konsantrasyonu 500 ppm dir. DİKKAT! 2-PE hızlı bir etkiye sahiptir. </li><li> Dolaşım sisteminden balık (AB zorlanma) istenen sayıda kaldırın. </li><li> 2 dakika (Şekil 1F) - 1 Anesteziyoloji içine balık aktarmak için bir net kullanın. 1 dakika içinde hızlı operkulum hareketlerini yavaş yavaş, yüzmek, yatay gasp göğüs yüzgeçleri yayıldı ve sahip balık gözlemleyin. </li><li> Zaman geçtikçe, balık kasanın altında yatıyordu gözlemlemek ve nihayet yüzme durdurun. Balık gasp durur ve operkulum hareketleri yavaş olduğunda anestezi cerrahi uçağı ulaşacak. Bu noktada, balık kan toplama için hazırdır. </li><li> Bir skimmer hafifçe 2-PE anestezi balık kaldırın ve kullanma anestezik (Şekil 1G) ile ıslatılmış kağıt havlu üzerine yerleştirin. Göz kuruluğunu önlemek ve yavaşça başka bir kuru ve yumuşak doku kağıt kullanmak da 2-PE çözeltisi ile ıslatılmış yumuşak doku kağıt ile balığın baş örtüsüvücut yüzeyinden kurulayın. </li></ol> 3. Kan Toplama <ol><li> Aspiratör tüp montajı (ya da ampul dağıtıcı) burunluğu sonunda bir heparinize iğne yerleştirin ve ağızda aspiratör borusu düzeneğinin ağızlık ucunu tutun. </li><li> Burunluk ucu bir arada iğne tutun ve dikkatlice iğne ucu ile müdahale ölçekleri kaldırın. Kan toplama sitesine 45 ° açı - 30 de iğne takın. Gastrointestinal sistem (Şekil 1H) bir patlağı kaçının. Yukarıda açıklandığı gibi ampul dağıtıcı kullanılması durumunda, başparmak ve orta parmak ile ampul basın ve ilk parmak ile ampulün ucundaki delik blok, sonra iğneyi. Not: Kan toplama sitesi dorsal aorta bölgesinde anüs vücut ekseni ve posterior boyunca olduğunu. Dorsal aorta (DA) ve posterior kardinal ven (PCV) omurga ventral sadece (Şekil 2). </li><li> Emmek başlayıniğne omurgayı dokunmadan hissettim zaman aspirasyon borusu düzeneğinin sonuna ağızlık. Kan yükselecek etmezse, kan akışını teşvik etmek için elle kurnazca iğne ucu hareket ettirin. Kan iğne içine yükseliyor kez hemen sallayarak durdurun ve yavaşça (Şekil 1i) emmek unutmayın. Ampul dağıtıcı kullanılması durumunda, kan aspire ampul basıncı boşaltın. </li><li> Kan yavaş nedeniyle arteriyel kan basıncının muhtemelen emme olmadan pulsatil bir tarzda iğne içine artacak gözlemleyin. Bu nedenle, iğne doğru arteri nüfuz halinde emmek için gerekli değildir. </li><li> Kanın uygun hacmi toplanır sonra emme durdurun. Balık iğneyi çıkarın ve herhangi bir kanamayı durdurmak için yumuşak doku kağıdı kullanarak ponksiyon sitesine basın. Parmak basıncı 20 sn (Şekil 1J) - yaklaşık 10 sonrası kanama durdurma gözlemleyin. </li><li> Kanama durdurulduktan sonra, derhal bir cle geri balık transferiBir sıcak su (~ 28 ° C) tankı. Yüzmek başlayana kadar yavaşça solungaçları doğru su dönen kurtarmak için balık yardımcı olur. Not: 2 L tankında 5 postsampling balık tutun ve balık oksijen sirkülasyon sistemine tankı bağlayın. Balık su antibiyotik ilave edilmesi gerekli değildir. Normal konut ve beslenme ile balık koruyun. </li><li> Parafilm bir parça (Şekil 1K) temiz bir alan üzerine iğne kan kovmak. </li><li> Herhangi bir ticari el glukometre (Şekil 1 L) kullanılarak kan şekeri ölçün. Not: Glucometer bir glikoz dehidrojenaz-flavin adenin dinükleotid elektrot kullanır ve 0,6 ul örnek bir hacimde kullanılmasını gerektirir. <ol><li> Metre içine tamamen bir test bandı takın ve doğrudan kan damlası dokunun. Otomatik olarak test şeridi içine kan beraberlik gözlemleyin ve görüntüleme alanında kan şekeri sonucu 5 sn edinin. Sonucu kaydedin ve test bandını atın. KullanınHer ölçüm için yeni bir test şeridi. </li></ol></li><li> (isteğe bağlı adım) bir pipetleme kanın doğru miktarda alın ve daha analizler (hemoglobin, triaçilgliserol (TG), total kolesterol, vs.) bir mikrosantrifüj tüp kan transferi. Gerekirse, tuzlu su ile bir balık tam kan seyreltin. RT&#39;de 680 x g&#39;de 3 dakika boyunca kan örnekleri santrifüj ve plazma hasat. Yeni bir tüp içine plazma aktarın. Bu noktada, bu biyokimyasal analizlerde kullanılmak üzere hazırdır. </li></ol>

<ol>
	<li>Lieschke, G., Currie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Penberthy, W. T., Shafizadeh, E., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Front Biosci. 7, d1439-d1453 (2002).</li><li>Stachura, D. L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dissection+of+zebrafish+hematopoiesis.">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 101, 75-110 (2011).</li><li>Jagadeeswaran, P., Sheehan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blood+coagulation+in+the+zebrafish.">Analysis of blood coagulation in the zebrafish.</a> Blood Cells Mol Dis. 25 (3-4), 239-249 (1999).</li><li>Jagadeeswaran, P., Sheehan, J. P., Craig, F. E., Troyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+zebrafish+thrombocytes.">Identification and characterization of zebrafish thrombocytes.</a> Br J Haematol. 107 (4), 731-738 (1999).</li><li>Lieschke, G. J., Oates, A. C., Crowhurst, M. O., Ward, A. C., Layton, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+functional+characterization+of+granulocytes+and+macrophages+in+embryonic+and+adult.">Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult.</a> Blood. 98 (10), 3087-3096 (2001).</li><li>Iwanami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+understanding+the+evolution+of+the+vertebrate+immune+system+and+human+primary+immunodeficiency.">Zebrafish as a model for understanding the evolution of the vertebrate immune system and human primary immunodeficiency.</a> Exp Hematol. 42 (8), 697-706 (2014).</li><li>Babin, P. J., Goizet, C., Raldua, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+of+human+motor+neuron+diseases:+advantages+and+limitations.">Zebrafish models of human motor neuron diseases: advantages and limitations.</a> Prog Neurobiol. 118, 36-58 (2014).</li><li>Seth, A., Stemple, D. L., Barroso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+use+of+zebrafish+to+model+metabolic+disease.">The emerging use of zebrafish to model metabolic disease.</a> Dis Mod Mech. 6 (5), 1080-1088 (2013).</li><li>Eames, S. C., Philipson, L. H., Prince, V. E., Kinkel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sugar+measurement+in+zebrafish+reveals+dynamics+of+glucose+homeostasis.">Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis.</a> Zebrafish. 7 (2), 205-213 (2010).</li><li>Moss, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+pancreas+in+adult+zebrafish.">Regeneration of the pancreas in adult zebrafish.</a> Diabetes. 58 (8), 1844-1851 (2009).</li><li>Oka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet-induced+obesity+in+zebrafish+shares+common+pathophysiological+pathways+with+mammalian+obesity.">Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.</a> BMC Physiol. 10 (21), (2010).</li><li>Chu, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Akt1+enhances+adipogenesis+and+leads+to+lipoma+formation+in+zebrafish.">Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish.</a> PLoS One. 7 (5), e36474 (2012).</li><li>Velasco-Santamar&#237;a, Y. M., Korsgaard, B., Madsen, S. S., Bjerregaard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bezafibrate,+a+lipid-lowering+pharmaceutical,+as+a+potential+endocrine+disruptor+in+male+zebrafish+(Danio+rerio).">Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (Danio rerio).</a> Aquat Toxicol. 105 (1-2), 107-118 (2011).</li><li>Zang, L., Shimada, Y., Nishimura, Y., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+reliable+method+for+repeated+blood+collection+from+aquarium+fish.">A novel, reliable method for repeated blood collection from aquarium fish.</a> Zebrafish. 10 (3), 425-432 (2013).</li><li>Carmichael, C., Westerfield, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+in+vitro+fertilization+at+the+zebrafish+international+resource+center.">Cryopreservation and in vitro fertilization at the zebrafish international resource center.</a> Methods Mol Biol. 546, 45-65 (2009).</li><li>Thorson, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+partitioning+of+body+water+in+Osteichthyes:+phylogenetic+and+ecological+implications+in+aquatic+vertebrates.">The partitioning of body water in Osteichthyes: phylogenetic and ecological implications in aquatic vertebrates.</a> Biol Bull-US. 120, 238-254 (1961).</li><li>Conte, F. P., Wagner, H. H., Harris, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+blood+volume+in+the+fish+(Salmo+gairdneri+gairdneri).">Measurement of blood volume in the fish (Salmo gairdneri gairdneri).</a> Am J Physiol. 205, 533-540 (1963).</li><li>Diehl, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> J Appl Toxicol. 21 (1), 15-23 (2001).</li><li>Nahas, K., Provost, J. -. P., Baneux, P. H., Rabemampianina, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+blood+removal+via+the+sublingual+vein+on+haematological+and+clinical+parameters+in+Sprague-Dawley+rats.">Effects of acute blood removal via the sublingual vein on haematological and clinical parameters in Sprague-Dawley rats.</a> Lab Anim. 34 (4), 362-371 (2000).</li><li>Curado, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+targeted+cell+ablation+in+zebrafish:+a+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> DevDyn. 236 (4), 1025-1035 (2007).</li><li>Andersson, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+signaling+promotes+regeneration+of+pancreatic+beta+cells+in+vivo.">Adenosine signaling promotes regeneration of pancreatic beta cells in vivo.</a> Cell Metab. 15 (6), 885-894 (2012).</li><li>Hiramitsu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eriocitrin+ameliorates+diet-induced+hepatic+steatosis+with+activation+of+mitochondrial+biogenesis.">Eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis.</a> Sci Rep-UK. 4, 3708 (2014).</li><li>Zang, L., Shimada, Y., Kawajiri, J., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Yuzu+(Citrus+junos+Siebold+ex+Tanaka)+peel+on+the+diet-induced+obesity+in+a+zebrafish+model.">Effects of Yuzu (Citrus junos Siebold ex Tanaka) peel on the diet-induced obesity in a zebrafish model.</a> J Funct Foods. 10, 499-510 (2014).</li><li>Schlegel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+non-alcoholic+fatty+liver+disease+with+zebrafish:+a+confluence+of+optics,+genetics,+and+physiology.">Studying non-alcoholic fatty liver disease with zebrafish: a confluence of optics, genetics, and physiology.</a> Cell Mol Life Sci. 69 (23), 3953-3961 (2012).</li><li>Stoletov, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+lipid+accumulation,+lipoprotein+oxidation,+and+macrophage+lipid+uptake+in+hypercholesterolemic+zebrafish.">Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.</a> Circ Res. 104 (8), 952-960 (2009).</li><li>Thomas, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+balance+and+energy+expenditure+during+ad+libitum+feeding+of+high-fat+and+high-carbohydrate+diets+in+humans.">Nutrient balance and energy expenditure during ad libitum feeding of high-fat and high-carbohydrate diets in humans.</a> Am J Clin Nutr. 55 (5), 934-942 (1992).</li></ol>

The authors have nothing to disclose.

Biz seri erişkin zebrabalıkları kan elde etmek için burada ayrıntılı bir protokol mevcut. Bu yöntem gerçekleştirmek için basittir ve günlük olarak laboratuvarda kullanın. Bu kan toplama yöntemi Zebra balığı en dorsal aort içine bir cam kılcal iğne takmadan dayanmaktadır. Bu işlem sırasında, o dorsal aorta arıyor için kriter olduğu için omurgayı baskılamak için dikkatli olmak önemlidir. Omurga yaralanması azaltılması hayatta kalma oranını artıracaktır. Bu teknik, basit ve usta kola...

Organları ve genetik insanlarda 1,2 benzemektedir, çünkü balığı insan hastalıklarında değerli bir model olarak artan bir popülerlik kazanmaktadır. Gelişimsel biyoloji alanında birçok çalışma zebra balığı ve insan gösteri hematopoiesis 3, hemostaz 4,5 ve myelopoiesis 6 benzerliği işaretli olduğunu göstermiştir. Yetişkin Zebra balığı da bu model organizma hisseleri nedeniyle insan hastalıklarında bozulduğu olanlarla ortak yollar immünolojik 7, nörodejeneratif 8 ve obezite ile ilişkili hastalıkların 9 çalışmak için kullanılır. Obezite ve obezite ile ilişkili hastalıkların (diyabet, karaciğer yağlanması ve alkolsüz steatohepatit ve ateroskleroz) için zebrabalıkları kan şekeri ve lipidler düzeyleri iyice birçok transgenik ve diyete bağlı obezite modellerinde 10-13 araştırılmıştır. Bireysel hayvanlardan Tekrarlanan kan örneklemesi hayvan kullanımı ve dec azaltacaktırbireyler farklılıkları artma. Ancak, tekrarlanan numune toplama nedeniyle nispeten küçük kan hacmi ve kolay erişilebilir damarların olmaması gibi zebrabalıkları gibi küçük hayvanlarda teknik olarak zordur. Bu yöntemler de dahil olmak üzere öldürücülüğü kendi dezavantajları, olmasına rağmen zebrafish bir defalık kan toplama için çeşitli yöntemler, ilişkili doku hasarı ve sınırlı kan hacmi geliştirilmiştir. Örneğin, 1-5 ul kan dorsal aorta 5 bölgesinde yaklaşık olarak boyuna 0.3 cm bir yanal kesi hasat edilebilir. Göğüs kuşak boyunca keserek makas ile Decapitation 5-10 ul kan 10 toplayabilir. Başka uygun kan örnekleme metodu kuyruk ablasyon 14&#39;tür. Kardiyak delinme aynı balık tekrarlanan kan toplama için tek potansiyel alternatif bir yöntemdir, ancak çok küçük bir miktar bu prosedüre (yaklaşık 50 nl) elde perfor olabilir analizlerin sayısını sınırlar11 med. Bu duruma göre, yeni bir protokol bu organizma insan hastalıkları için standart model organizma için önemli bir ilerleme gerekli olacaktır tekrarlanan öldürücü olmayan kan örnekleme sağlamak için gereklidir. Bu teknik farmakolojik cevabı, moleküler tanı için biyobelirteçlerin, prognoz tayini keşif ve metabolik hastalıklar, dejeneratif hastalıklar ve maligniteler çeşitli türleri gibi çeşitli hastalıkların izlenmesi test etmek için izin verecek. Bu nedenle Zebra balığı seri 15 kan elde etmek için minimal invaziv bir yöntem geliştirdi. Burada görsel prosedür göstermek ve bu teknik için ayrıntılı bir protokol sağlar. Bu yöntemi kullanarak, çeşitli hemoglobin içeren parametreler, açlık kan şekeri, sağlıklı erişkin zebrabalıkları kanında lipidler dayalı normal değer değerlendirildi. Ayrıca, aynı zamanda, bu yöntem, m seri örnekleri gerektiren çalışmalar için uygun olup olmadığını değerlendiriroverfeeding deneyler sırasında kan glukoz düzeylerinde geçici değişiklikler onitoring.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass capillaries with filament</td>
			<td>Narishige</td>
			<td>GD-1</td>
			<td>1.0-mm-outer-diameter.</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>To produce the needls</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Wako Pure Chemical Industries</td>
			<td>081-00136</td>
			<td>For heparinization</td>
		</tr>
		<tr>
			<td>Aspirator tube assembly</td>
			<td>Drummond</td>
			<td>2-040-000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>Bulb dispenser</td>
			<td>Drummond</td>
			<td>1-000-9000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>2-phenoxyethanol</td>
			<td>Wako Pure Chemical Industries</td>
			<td>163-12075</td>
			<td>For anesthetizing the fish</td>
		</tr>
		<tr>
			<td>DRI-CHEM3500V</td>
			<td>Fujifilm</td>
			<td>-</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>DRI-CHEM Slides</td>
			<td>Fujifilm</td>
			<td>Hb-WII</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>Glutest Neo Super</td>
			<td>Sanwa Kagaku Kenkyusho</td>
			<td>-</td>
			<td>For bood glucose measurement</td>
		</tr>
		<tr>
			<td>Wako L-type TG kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>464-44201</td>
			<td>For TG measurement</td>
		</tr>
		<tr>
			<td>Wako L-type CHO kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>460-44301</td>
			<td>For total cholesterol measurement</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Alcan Packaging</td>
			<td>PM996</td>
			<td>To expel the blood on</td>
		</tr>
	</tbody>
</table>

repeated blood collection for blood tests in adult zebrafish

Tekrarlanan kan toplama laboratuvar hayvanları üzerinde yapılan en yaygın tekniklerden biridir. Ancak, Zebra balığı kan toplama için bir öldürücü olmayan protokol kurulmamıştır. Zebra balığı kan toplama için önceki metotlar, lateral insizyon, dekapitasyon ve kuyruk ablasyon gibi öldürücü bulunmaktadır. Böylece biz burada bu yordamı özetleyen ayrıntılı bir protokol, bir roman &quot;tekrarlanan&quot; kan toplama yöntemi geliştirdi ve mevcut var. Bu yöntem, minimal invaziv ve bu nedenle, aynı bireyden elde tekrarlanan kan örnekleme sağlayan zebrabalıkları için çok düşük bir ölüm oranına (% 2.3) elde edilir. Kan örneği maksimum hacim balık vücut ağırlığına bağlıdır. Aralıklarla tekrarlanan kan örneklemesi için hacim kan hemoglobin ölçümleri ile değerlendirildi vücut ağırlığının her hafta ya da ≤1% 2 haftada, bir ≤0.4% olmalıdır. Ayrıca, hemoglobin, açlık kan şekeri, plazma triaçilgliserol (TG) ve toplam cerkek ve dişi yetişkin zebrafish holesterol düzeyleri ölçüldü. Ayrıca diyet indükte obeziteye glikoz metabolizmasının bozulmasına araştırmak için, bu yöntemi uygulanır. Bu kan toplama yöntemi glukoz ve lipit metabolizması ve bir insan hastalık model organizma olarak zebrafish kullanımını artıracak hematolojik çalışmalar da dahil olmak üzere birçok uygulama, sağlayacaktır.

(Şekil 1J &lt;1 mm delinmesi) ve% 2.3 gibi çok düşük bir mortalite oranı veren bu kan toplama yöntemi zebrabalıkları minimal hasara neden olmaktadır. Tek bir balık toplanan ve vücut ağırlığı (Şekil 3) bir ilişki değerlendirilebilir kan maksimum ses incelenmiştir. Biz toplanan maksimum kan hacmi doğrusal vücut ağırlığının (R = 0,813) ile ilişkili olduğunu bulduk. Tek bir balık (vücut ağırlığı = 1,071 g) toplanan kanın en büyük hacmi 25 ul oldu ve ...

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

Repeated Blood Collection for Blood Tests in Adult Zebrafish

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

Yetişkin Zebra balığı Kan Testleri için Tekrarlanan Kan Alma

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

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.5K Görüntüleme.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Video: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Amerikan Horseshoe Yengeç, Limulus Polyphemus&#39;un Kan Toplama

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Biyoloji

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Yenidoğan Fareler Pulmoner Endotel Hücreleri İzolasyon ve Kültür

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Embriyonik Ekstremite Cilt damarları Tüm montaj İmmünohistokimyasal Analizi: Embriyo vasküler dallanma Morphogenesis Eğitim Model Sistemi

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Yetişkin Zebra balığı Biyokimyasal Analiz için Kan Alma

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

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 ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Kinaz İnhibitörleri Karşı Dayanıklı Mutasyonlarının Tarama ve Doğrulama için Bir Yöntem

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Akım Sitometri Kullanımı Ekstrasellüler Veziküller Analizi Teknikleri

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

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

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