Yazarlar Hammond Bay Biyolojik İstasyonu, Great Lakes Science Center, US Geological Survey katkısını kabul. Biz de lazer tarama konfokal mikroskopi onun teknik destek için Michigan State Üniversitesi İleri Mikroskopi Merkezi&#39;nde Dr Melinda Frame teşekkür ederim. Bu çalışma YWCD ve WML Büyük Göller Balıkçılık Komisyonu hibe tarafından desteklenmektedir.

1.. Çözüm Hazırlığı <ol><li> 1 L sağlayın 10x 0.1 M fosfat tampon tuzlu su (PBS, pH 7.4): 26.2 gr sodyum fosfat (monobazik), 115 g sodyum fosfat (dibazik), 87.66 g NaCI ve tartılır. Yaklaşık 800 ml distile H 2 O içinde çözülür, pH ayarlanması, ve distile H 2 O. ile 1 L&#39;ye hacmi getirmek <ol><li> 1 L 0.1 M fosfat tampon tuzlu su (pH 7.4) Marka: 100 ml 10x PBS alın ve 900 ml damıtık H2O eklemek </li><li> 100 ml 10x PBS al, 1 ml Triton X-100 ekleyin ve damıtık H2O ile 1 L&#39;ye getir hacim: 1 L 0.1 M fosfat tampon tuzlu su (pH 7.4),% 0.1 Triton X-100 ile yapın </li></ol></li><li> 26.2 g sodyum fosfat (monobazik) ve 115 g sodyum fosfat (dibazik) tartılır: 1 L 10x 0.1 M fosfat tamponu (PB, pH 7.4) olun. Yaklaşık 800 ml distile H 2 O içinde çözülür, pH ayarlanması, ve distile H 2 O. ile 1 L&#39;ye hacmi getirmek <ol><li> 1 L 0.1 M fosfat tamponu (pH 7.4) Marka: Al 100 ml 10x PB,ve H 2 O. damıtılmış 900 ml ekle </li><li> % 0.1 Triton X-100 ile 1 L 0.1 M fosfat tamponu (pH 7.4) Marka: 100 ml 10x PB al, 1 ml Triton X-100 ekleyin ve damıtık H2O ile 1 L&#39;ye kadar ses getirmek </li></ol></li><li> 400 ml hidrojel monomer çözeltisi (% 4 akrilamit,% 0.25 bis-akrilamid,% 4 paraformaldehit,% 0.0075 amonyum persülfat, 0.1 M PBS içinde% 0.0005 saponin) Marka: 0.3 g amonyum persülfat ve 0.2 g saponin tartılır. 210 ml damıtılmış 2 H ekleme O, 40 ml% 40 akrilamit,% 2 10 mi bis-akrilamid, 40 ml 10x PBS (pH 7.4), 100 ml% 16 paraformaldehit ve iyice karıştırın. </li><li> (0.2 M borik asit,% 4 SDS) 4 L temizleme çözeltisi oluşturunuz: SDS g 49,464 g borik asit ve 160 tartılır. , Yaklaşık 3,5 L distile H 2 O eritin NaOH ile 8.5 pH ayarlamak ve 4 L. hacmi getirmek </li><li> 1 L% 80 gliserol çözeltisi Marka:, 800 ml gliserol ölçmek 200 ml damıtık H2O ekleyin ve iyice karıştırın. </li></ol> 2.. Doku Hazırlanması <ol><li> Her bir 15 ml santrifüj tüpüne hidrojel monomer çözeltisi ve kısım 10 mi hazırlayın. </li><li> Bütün doku inceleyin ve 4 ° C&#39;de 2 gün için hidrojel saptamak Hidrojel çözelti en az 10x doku hacmi olduğundan emin olun. </li><li> Bir çeker ocak için (15 ml santrifüj tüpüne) hidrojel sabit doku getirin. </li><li> , TEMED 5 ul ekleyerek hidrojel sabit doku polimerize iyice karıştırın ve 2-3 saat boyunca oda sıcaklığında, çeker ocak içinde bekletin. </li><li> İyice fazla jelin çıkarılması. Not: Ekstra jel boyama esnasında temizleme sürecini ve antikor sokulmasına engel olacaktır. </li><li> Doku saydam hale gelinceye kadar, birkaç gün boyunca 70 rpm&#39;de ve 50 ° C&#39;de ayarlanmış bir kuluçka çalkalayıcı içinde çözelti temizleme 10 ml sabit doku inkübe edin. , En az günde bir kez temizleme çözeltisi değiştirmek ve temizleme solüsyonu değiştirirken dokudan fazla jelin çıkarılması. </li><li> 70 r% 0.1 Triton X-100 ile 10 ml 0.1 M PB içerisinde dokuyu durulayınpm ve 37 ° C gecede. Bu adım 3 kez tekrarlayın ve tampon çözeltisi değiştirirken dokudan fazla jelin çıkarılması. </li><li> 2 gün boyunca 70 rpm ve 37 ° C &#39;de% 0.1 Triton X-100 ile PB primer antikor çözeltisi içindeki açıklık dokuyu inkübe edin. Not: Bütün daldırın doku için yeterli birincil antikor çözüm olun. </li><li> Gece boyunca 70 rpm ve 37 ° C &#39;de% 0.1 Triton X-100 ile 10 ml PB doku durulayın. Bu adım 3 kez tekrarlayın ve tampon çözeltisi değiştirirken dokudan fazla jelin çıkarılması. </li><li> Karanlık bir odada ya da loş ışık altında, aşağıdaki adımları uygulayın. Fotoğraf beyazlatma önlemek için folyo ile örtün örnekleri. </li><li> % 0.1 Triton X-100 ile PB floresan ikincil antikor çözeltisi ve 1 gün boyunca 70 rpm ve 37 ° C de bloke serumu ile berraklaştırılmış dokuyu inkübe edin. </li><li> Gece boyunca 70 rpm ve 37 ° C &#39;de% 0.1 Triton X-100 ile 10 ml PB doku durulayın. Bu adımı 3 kez tekrarlayın ve aşırı jel f kaldırmakTampon çözeltisi değiştirirken doku rom. </li><li> Gece boyunca 70 rpm ve 37 ° C de 10 ml PBS içinde doku durulayın. Bu adım 3 kez tekrarlayın ve tampon çözeltisi değiştirirken dokudan fazla jelin çıkarılması. </li><li> 70 rpm ve 37 ° C sıcaklıkta gece boyunca% 80 gliserol içinde berraklaştırılmış doku inkübe edin. </li><li> Önce konfokal mikroskopi için 4 ° C&#39;de floresan lekeli doku saklayın. </li></ol>

<ol>
	<li>Applegate, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+the+sea+lamprey+(Petromyzon+marinus)+in+Michigan.">Natural history of the sea lamprey (Petromyzon marinus) in Michigan.</a> US Fish and Wildlife Service Special Science Report on Fishery Service. (55), (1950).</li><li>Hardisty, M. W., Potter, I. C., Hardisty, M. W., Potter, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+general+biology+of+adult+lampreys.">The general biology of adult lampreys.</a> The biology of lampreys. 1, 127-206 (1971).</li><li>Youson, J. H., Potter, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+description+of+the+stages+in+the+metamorphosis+of+the+anadromous+sea+lamprey,+Petromyzon+marinus+L.">A description of the stages in the metamorphosis of the anadromous sea lamprey, Petromyzon marinus L.</a> Can. J. Zool. 57, 1808-1817 (1979).</li><li>Boomer, L. A., 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=Cholangiocyte+apoptosis+is+an+early+event+during+induced+metamorphosis+in+the+sea+lamprey,+Petromyzon+marinus+L.">Cholangiocyte apoptosis is an early event during induced metamorphosis in the sea lamprey, Petromyzon marinus L.</a> J. Pediatr. Surg. 45, 114-120 (2010).</li><li>Kasai, M., Suzuki, H., Ohashi, E., Ohi, R., Chiba, T., Okamoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+and+results+of+operative+management+of+biliary+atresia.">Technique and results of operative management of biliary atresia.</a> World J. Surg. 2, 571-580 (1978).</li><li>Suzuki, T., Hashimoto, T., Kondo, S., Sato, Y., Hussein, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+patients&#8217;+outcome+post-Kasai+operation:+a+19-year+experience+with+modification+of+the+hepatic+portoenterostomy+and+applying+a+novel+steroid+therapy+regimen.">Evaluating patients&#8217; outcome post-Kasai operation: a 19-year experience with modification of the hepatic portoenterostomy and applying a novel steroid therapy regimen.</a> Pediatr. Surg. Int. 26, 825-830 (2010).</li><li>Hartley, J. L., Davenport, M., Kelly, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia.">Biliary atresia.</a> Lancet. 374, 1704-1713 (2009).</li><li>Morecki, R., Glaser, J. H., Cho, S., Balistreri, W. F., Horwitz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia+and+reovirus+type+3+infection.">Biliary atresia and reovirus type 3 infection.</a> New Engl. J. Med. 307, 481-484 (1982).</li><li>Shimadera, S., Iwai, N., Deguchi, E., Kimura, O., Fumino, S., Yokoyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inv+mouse+as+an+experimental+model+of+biliary+atresia.">The inv mouse as an experimental model of biliary atresia.</a> J. Pediatr. Surg. 42, 1555-1560 (2007).</li><li>Sidon, E. W., Youson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+changes+in+the+liver+of+the+sea+lamprey,+Petromyzon+marinus+L.,+during+metamorphosis:+I.+Atresia+of+the+bile+ducts.">Morphological changes in the liver of the sea lamprey, Petromyzon marinus L., during metamorphosis: I. Atresia of the bile ducts.</a> J. Morphol. 177, 109-124 (1983).</li><li>Yeh, C. -. Y., Chung-Davidson, Y. -. W., Wang, H., Li, K., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+synthesis+and+secretion+of+bile+salts+as+an+adaptation+to+developmental+biliary+atresia+in+the+sea+lamprey.">Intestinal synthesis and secretion of bile salts as an adaptation to developmental biliary atresia in the sea lamprey.</a> Proc. Nat. Acad. Sci. U.S.A. 109, 11419-11424 (2012).</li><li>Chung, 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497, 332-337 (2013).</li><li>Alarc&#243;n, V. B., Filosa, M. F., Youson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokeratins+in+the+liver+of+the+sea+lamprey+(Petromyzon+marinus)+before+and+after+metamorphosis.">Cytokeratins in the liver of the sea lamprey (Petromyzon marinus) before and after metamorphosis.</a> Cell Tissue Res. 287, 365-374 (1997).</li><li>Youson, J. H., Ogilvie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+features+of+degeneration+of+the+gallbladder+during+lamprey+biliary+atresia.">Ultrastructural features of degeneration of the gallbladder during lamprey biliary atresia.</a> Tissue and Cell. 22, 477-492 (1990).</li><li>Morii, 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=Onset+of+apoptosis+in+the+cystic+duct+during+metamorphosis+of+a+Japanese+lamprey,+Lethenteron+reissneri.">Onset of apoptosis in the cystic duct during metamorphosis of a Japanese lamprey, Lethenteron reissneri.</a> Anat. Rec. 293, 1155-1166 (2010).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Bu protokol, nano gözenekli bir hidrojel oluşturmak üzere, poliakrilamid ile bozulmamış doku çapraz bağlar ve daha sonra optik saydamlık ve makromoleküler geçirgenliği elde etmek için doku plazma zarı uzak şeritler &quot;açıklık&quot; 12 olarak adlandırılan yeni bir yöntem, modifiye edilir. &quot;BERRAKLIK&quot; uzun menzilli projeksiyon ve sinir sisteminde yerel devre kablolama sağlam doku görüntüleme sağlar. Bu yeni yöntem, başkalaşım sırasında deniz lamprey karaciğerde tüm ...

Deniz taşemen üç ayrı yaşam boyunca gelişir 1,2 aşamaları. Larva deniz taşemen (L) bentik filtre besleyiciler olarak yuvalarda çoğu zaman harcamak. Dış morfoloji ve iç organlarda 3 yeniden dramatik değişiklikler yedi metamorfik aşamaları geçtikten sonra, elde edilen yavrular (JV) kütle 100 defadan fazla vücut artırarak, konak balık kan ve doku sıvıları ile beslenirler sırasında bir parazit sahne girin . Okyanus veya büyük göllerde konak balık besleme, 1.0 ila 1.5 yıl sonra, yetişkin erken bahar aylarında beslenme durdurma ve yumurtlamak için akarsulara göç ve ardından 1,2 ölür. Metamorfoz sırasında, deniz taşemen karaciğer safra kesesi ve safra yolları, tüm, insan bebekte hastalık safra yolları atrezisi taklit eden evrimsel bir mutant fenotip kaybeder. Bebek biliyer atrezi 4,5,6,7 ciddi tıbbi komplikasyonlar nadir bir pediatrik karaciğer hastalığıdır, 8,9,10, ancak safra atrezi patogenezi ve etyolojisi 4 büyük ölçüde bilinmemektedir. Cerrahi müdahale (Kasai prosedürü) 5 gerçekleştirilir sürece biliyer atrezi ile hastalar doğumdan sonra iki yıl içinde ölmektedir. Daha sonra, bu hastalar geniş klinik yönetimi ve genellikle karaciğer nakli 6. gerektirir. Biliyer atrezi etiyopatogenezinin Birçok teori gibi viral enfeksiyon, konjenital malformasyon, otoimmün hastalık, toksik ve hakaret olarak önerilmiştir. Ancak, safra atrezi gelişimine her katkısı 7,8,9,10 sonuçsuz kalır. Patolojik safra yolları atrezisi acı bebeklerin aksine, deniz taşemen gelişimsel kapsamlı stres nekroinflamasyon, fibrozis veya siroz 10 olmadan safra yolları atrezisi programlanmış geçireceği. Hayvanlar, bu süreçte 10 sırasında geçici kolestazınız acı, ama bu gelişimsel duruma adapte olabilirile de novo sentezi ve bu 11 karaciğer safra tuzunun sentezi azaltma olarak bilinen mekanizmaları ek olarak gelişim safra atrezi, sonra bağırsak safra tuzlarının salgılanması. Deniz lamprey Bu gelişim süreci biliyer atrezi ilerlemesini incelemek için bilinen tek fırsat sağlar. &quot;AÇIKLIK&quot; adı verilen yeni geliştirilmiş bir yöntem, bir optik şeffaf Nanogeçirgen hidrojel 12 içine bozulmamış dokusu dönüştürerek karmaşık memeli sinir sistemlerinde yüksek çözünürlüklü görüntüleme sağlar. Deniz taşemen karaciğer ve modifiye BERRAKLIK protokolünü kullanarak, safra dejenerasyon sağlam doku görüntüleme karaciğer metamorfoz boyunca belgelenmiştir olabilir.

<table border="0" cellpadding="0" cellspacing="0" width="702">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40% acrylamide&#160;</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:119px;">161-0140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2% bis-acrylamide&#160;</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammonium persulfate&#160;</td>
			<td>Sigma</td>
			<td>A3678-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">boric acid</td>
			<td>Sigma</td>
			<td>B7901-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">saponin&#160;</td>
			<td>Sigma</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium dodecyl sulfate&#160;</td>
			<td>Sigma</td>
			<td style="width:119px;">L337-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate (monobasic)</td>
			<td>Sigma</td>
			<td>04269-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate (dibasic)</td>
			<td>Sigma</td>
			<td>S5136-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol&#160;</td>
			<td>Sigma</td>
			<td>G9012-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH pellets&#160;</td>
			<td>EMD</td>
			<td>SX0590-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml centrifuge tubes</td>
			<td style="width:200px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting tools&#160;</td>
			<td style="width:200px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a new clarification method to visualize biliary degeneration during liver metamorphosis in sea lamprey (petromyzon marinus)

Biliyer atrezi Tayvan 5,000 içinde 1 bildirilen frekans ile tahmini 1 15,000 güneydoğu ABD&#39;de frekans, ancak Doğu Asya ülkelerinde daha yaygın olan, bebeklik nadir bir hastalıktır. Çok biliyer atrezi yönetimi hakkında bilinmesine rağmen, patogenezi hala zor olduğunu. Deniz taşemen (Petromyzon marinus) safra dejenerasyonun mekanizması ve ilerlemesini incelemek için eşsiz bir fırsat sunmaktadır. Larva, parazit ve yetişkin: Deniz taşemen üç farklı yaşam evrelerinde yoluyla gelişir. Larva parazitik koleje geçiş sırasında, deniz taşemen dış morfolojisi ve iç organlarda dramatik yeniden yapılanma ve yeniden birlikte metamorfoz tabi. Karaciğerde, tüm bilier sistem safra kesesi ve safra dahil, kaybolur. &quot;AÇIKLIK&quot; adı verilen yeni geliştirilmiş bir yöntem metamorfik deniz lamprey de bağırsak ile tüm karaciğer ve kavşak netleştirmek için güncellenmiştir.safra dejenerasyon süreci lazer tarama konfokal mikroskopi kullanılarak görüntülendi ve deniz taşemen metamorfoz sırasında ayırt edildi. Bu yöntem, eşsiz bir hayvan modelinde safra yolları atrezisi incelemek için güçlü bir araç sağlar.

Birçok önemli gelişme olayları deniz taşemen Başkalaşım sırasında Hepatobilier sistemde meydana. Safra kanalı ve safra kesesi apoptozu ve dejenere (Şekil 1) tabi. Ve konfokal mikroskopi kullanılarak anti-apoptotik işaretleyici BCL2 (cholangiocytes ve hepatositler önce ve başkalaşım 13 sonrasında mevcut CK19) karaciğer hücre işaretleyici sitokeratin 19, modifiye arıtma yöntemi ve boyama birleştiren, tüm safra sistemi Z-ekseni (boyunca yakalandı Şekil 2)</str...

Watch this Scientific Journal Video about A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus at JoVE.com

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Deniz taşemen Başkalaşım sırasında safra kesesi ve safra yolları, insan safra atrezi ile benzer bir süreç kaybedersiniz. Yeni bir tespit ve açıklama yöntemi (AÇIKLIK) lazer tarama konfokal mikroskopi kullanılarak tüm safra yolları görselleştirmek için güncellenmiştir. Bu yöntem safra dejenerasyonu incelemek için güçlü bir araç sağlar.

Deniz taşemen Karaciğer Metamorfoz sırasında Safra Hastalığı gözünüzde Yeni Bir Açıklama Yöntemi ( Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

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Biology

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

13.7K Görüntüleme.  Michigan State University.  Deniz taşemen Başkalaşım sırasında safra kesesi ve safra yolları, insan safra atrezi ile benzer bir süreç kaybedersiniz. Yeni bir tespit ve açıklama yöntemi (AÇIKLIK) lazer tarama konfokal mikroskopi kullanılarak tüm safra yolları görselleştirmek için güncellenmiştir. Bu yöntem safra dejenerasyonu incelemek için güçlü bir araç sağlar. 

Video: A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Doğru Genotiplendirme için Basit Bir Yöntem

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Biyoloji

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

In Oksijen yetersizliği kaynaklı Asma Animasyon Visualize&#39;a Time Lapse Mikroskopi kullanın<em&gt; C. elegans</em&gt; Embriyolar

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

3D Nükleer teşkilatında değişiklikler Eğitim için 3D-korunmuş Tane Çekirdekle ilgili Kombine İmmünofloresan ve DNA FISH

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

Deniz Kestanesi Zigotlar Yüksek Randımanlı Microinjections

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

Squid pigmentlerin çıkarılması için bir yöntem<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

Safra Yolunun İnternalasyonunu İncelemek İçin Bir Tam Montajlı İmmünohistokimyasal Yöntem Kullanmak<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Suyu Microdilution Vitro tarama: yeni mantar önleyici bileşikler algılamak için kolay ve hızlı bir yöntem

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Split yeşil flüoresan Protein sistemi effectors görselleştirmek için bakteri enfeksiyonu sırasında teslim.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Deniz Hıyar Bağırsak Hücrelerinin Birincil Kültüründe Apoptosis İndüksiyon uyupve tayini

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...