Gli autori riconoscono il contributo della stazione Hammond Bay biologica, Great Lakes Science Center, US Geological Survey. Ringraziamo anche il Dott. Melinda frame presso il Centro di Microscopia Avanzata presso la Michigan State University per il suo supporto tecnico nella scansione laser microscopia confocale. Questo studio è sostenuto da finanziamenti della Commissione Grandi Laghi per la pesca a YWCD e WML.

1. Preparazione Solution <ol><li> Rendere 1 L 10x 0.1 M salina tampone fosfato (PBS, pH 7,4): Pesare 26,2 g di fosfato di sodio (monobasico), 115 g di fosfato di sodio (bibasico), e 87,66 g NaCl. Sciogliere in circa 800 ml di acqua distillata H 2 O, regolare il pH, e portare al volume di 1 litro con acqua distillata H 2 O. <ol><li> Rendere 1 L 0.1 M tampone fosfato salino (pH 7.4): Prendete 100 ml 10x PBS e aggiungere 900 ml di acqua distillata H 2 O. </li><li> Rendere 1 L 0.1 M tampone fosfato salino (pH 7.4) con 0.1% Triton X-100: Prendete 100 ml 10x PBS, aggiungere 1 ml di Triton X-100, e portare al volume di 1 litro con acqua distillata H 2 O. </li></ol></li><li> Rendere 1 L 10x 0.1M tampone fosfato (PB, pH 7,4): Pesare 26,2 g di fosfato di sodio (monobasico) e 115 g di fosfato di sodio (bibasico). Sciogliere in circa 800 ml di acqua distillata H 2 O, regolare il pH, e portare al volume di 1 litro con acqua distillata H 2 O. <ol><li> Rendere 1 L 0.1 M tampone fosfato (pH 7.4): Prendete 100 ml 10x PB,e aggiungere 900 ml di acqua distillata H 2 O. </li><li> Rendere 1 L 0.1 M tampone fosfato (pH 7.4) con 0.1% Triton X-100: Prendete 100 ml 10x PB, aggiungere 1 ml di Triton X-100, e portare al volume di 1 litro con acqua distillata H 2 O. </li></ol></li><li> Preparare la soluzione di 400 ml di idrogel monomero (4% acrilammide, 0,25% bis-acrilammide, 4% paraformaldeide, 0,0075% persolfato di ammonio, 0,0005% saponina in 0.1 M PBS): Pesare 0,3 g di persolfato di ammonio e 0,2 g saponina. Aggiungere 210 ml di H 2 O distillata, 40 ml 40% di acrilammide, 10 ml 2% bis-acrilammide, 40 ml 10x PBS (pH 7.4), 100 ml 16% paraformaldeide, e mescolare bene. </li><li> Preparare la soluzione di compensazione 4 L (4% SDS in acido borico 0.2 M): Pesare 49,464 g di acido borico e 160 g SDS. Sciogliere in circa 3,5 L H 2 O distillata, aggiustare il pH a 8.5 con NaOH, e portare il volume fino a 4 L. </li><li> Rendere 1 L soluzione di glicerolo 80%: Misurare 800 ml di glicerolo, aggiungere 200 ml di acqua distillata H 2 O, e mescolare bene. </li></ol> 2. Preparazione Tissue <ol><li> Preparare la soluzione di monomero idrogel e un&#39;aliquota di 10 ml per ogni provetta da centrifuga 15 ml. </li><li> Sezionare tutto il tessuto e fissare l&#39;idrogel per 2 giorni a 4 ° C. Assicurarsi che la soluzione idrogel è almeno 10 volte il volume del tessuto. </li><li> Portare il tessuto fissato idrogel (nella provetta da centrifuga 15 ml) ad una cappa aspirante. </li><li> Polimerizzare il tessuto fissato idrogel con l&#39;aggiunta di 5 ml TEMED, mescolare bene e lasciar riposare nella cappa a temperatura ambiente per 2-3 ore. </li><li> Rimuovere accuratamente il gel in eccesso. Nota: gel Extra ostacolerà il processo di compensazione e la penetrazione degli anticorpi durante la colorazione. </li><li> Incubare il tessuto fissato in 10 ml di soluzione di compensazione in un agitatore incubatore a 70 rpm e 50 ° C per diversi giorni finché il tessuto diventa trasparente. Cambiare soluzione compensazione almeno una volta al giorno e rimuovere il gel in eccesso dal tessuto quando si cambia la soluzione di compensazione. </li><li> Risciacquare il tessuto in 10 ml di 0,1 M PB con 0.1% Triton X-100 a 70 rpm e 37 ° C durante la notte. Ripetere questo passaggio 3 volte e rimuovere il gel in eccesso dal tessuto quando si cambia la soluzione tampone. </li><li> Incubare il tessuto chiarito nella soluzione di anticorpo primario in PB con 0.1% Triton X-100 a 70 rpm e 37 ° C per 2 giorni. Nota: soluzione di anticorpo primario sufficiente a sommergere tutto il tessuto. </li><li> Risciacquare il tessuto in 10 ml PB con 0.1% Triton X-100 a 70 rpm e 37 ° C durante la notte. Ripetere questo passaggio 3 volte e rimuovere il gel in eccesso dal tessuto quando si cambia la soluzione tampone. </li><li> Effettuare le seguenti operazioni in una stanza buia o sotto la luce fioca. Coprire i campioni con un foglio per evitare foto sbiancamento. </li><li> Incubare il tessuto chiarito con la soluzione di anticorpo secondario fluorescente in PB con 0.1% Triton X-100 e il siero di blocco a 70 rpm e 37 ° C per 1 giorni. </li><li> Risciacquare il tessuto in 10 ml PB con 0.1% Triton X-100 a 70 rpm e 37 ° C durante la notte. Ripetere questa operazione 3 volte e rimuovere il gel in eccesso fROM il tessuto quando si cambia la soluzione tampone. </li><li> Risciacquare il tessuto in 10 ml di PBS a 70 rpm e 37 ° C durante la notte. Ripetere questo passaggio 3 volte e rimuovere il gel in eccesso dal tessuto quando si cambia la soluzione tampone. </li><li> Incubare il tessuto chiarito in 80% glicerolo a 70 rpm e 37 ° C durante la notte. </li><li> Conservare il tessuto tinto fluorescente a 4 ° C prima di microscopia confocale. </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>

Gli autori non hanno nulla da rivelare.

Questo protocollo è modificato da un nuovo metodo chiamato &quot;chiarezza&quot; 12, che reticola tessuto intatto con poliacrilammide per formare un idrogel nanoporoso, e poi strappa via la membrana plasmatica del tessuto per ottenere la trasparenza ottica e permeabilità macromolecolare. &quot;CHIAREZZA&quot; permette l&#39;imaging intatto tessuto di lungo raggio di proiezione e il cablaggio del circuito locale nel sistema nervoso. Questo nuovo metodo può essere usato per visualizzare l&#39;intero sistema ...

Lampreda di mare si sviluppano attraverso tre distinte fasi di vita di 1,2. Larvale lampreda di mare (L) trascorrono più tempo in tane come filtratori bentonici. Dopo aver attraversato sette stadi metamorfici di drammatici cambiamenti nella morfologia e riorganizzazione in organi interni 3 esterni, il novellame risultanti (JV) entrano in una fase parassitaria nel corso della quale si nutrono di sangue e fluidi tissutali di pesce ospitante, l&#39;aumento della massa corporea più di 100 volte . Dopo la 1.0 to 1,5 anni di alimentazione sul pesce ospite in mare o grandi laghi, adulti cessano alimentazione durante la primavera e migrano nei fiumi per deporre le uova e poi muoiono 1,2. Durante la metamorfosi, il fegato lampreda di mare perde la cistifellea e l&#39;intero albero biliare, un fenotipo mutante evolutivo che imita la malattia neonato umano atresia delle vie biliari. Infant atresia biliare è una rara malattia del fegato pediatrico a gravi complicazioni mediche 4,5,6,7, 8,9,10, ma la patogenesi ed eziologia di atresia biliare sono in gran parte sconosciuti 4. I pazienti con atresia biliare muoiono entro due anni dopo la nascita se non viene eseguito l&#39;intervento chirurgico (procedura Kasai) 5. Successivamente, questi pazienti richiedono una gestione clinica e spesso trapianto di fegato 6. Molte teorie di atresia biliare eziopatogenesi sono stati proposti, come l&#39;infezione virale, malformazione congenita, malattia autoimmune, e insulto tossico. Tuttavia, il contributo di ciascuno allo sviluppo di atresia biliare rimane inconcludente 7,8,9,10. A differenza dei bambini che soffrono patologico atresia delle vie biliari, lampreda di mare che formano oggetto evolutivamente programmato atresia delle vie biliari senza una necroinfiammazione, fibrosi o cirrosi 10. Gli animali possono soffrire colestasi transitorio durante questo processo 10, ma adattarsi a questa condizione dello sviluppovia de novo sintesi e secrezione di sali biliari nell&#39;intestino dopo sviluppo atresia biliare, oltre ai meccanismi noti come la riduzione della sintesi di sali biliari nel fegato 11. Questo processo di sviluppo in lampreda di mare offre l&#39;opportunità unica nota di esaminare la progressione di atresia biliare. Un metodo di nuova concezione denominato &quot;CHIAREZZA&quot; permette di imaging ad alta risoluzione in complessi sistemi nervosi dei mammiferi trasformando tessuto intatto in un idrogel nanoporosa otticamente trasparente 12. Utilizzando mare fegato lampreda e un protocollo CLARITY modificato, imaging intatto tessuto della degenerazione biliare può essere documentata in tutta metamorfosi fegato.

<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)

Atresia biliare è una rara malattia dell&#39;infanzia, con una stima di 1 a 15.000 frequenza nel sud-est degli Stati Uniti, ma più comune nei paesi dell&#39;Asia orientale, con una frequenza riportata di 1 su 5.000 in Taiwan. Anche se si sa molto circa la gestione di atresia biliare, la sua patogenesi è ancora sfuggente. La lampreda di mare (Petromyzon marinus) offre un&#39;opportunità unica per esaminare il meccanismo e la progressione della degenerazione biliare. Lampreda di mare sviluppare attraverso tre fasi della vita distinte: larve, parassiti, e adulti. Durante la transizione da larve di parassiti giovanile, lampreda di mare subiscono metamorfosi con la riorganizzazione drammatica e rimodellamento in morfologia esterna e gli organi interni. Nel fegato, l&#39;intero sistema biliare è perso, compresa la cistifellea e l&#39;albero biliare. Un metodo di nuova concezione denominato &quot;CHIAREZZA&quot; è stata modificata per chiarire l&#39;intero fegato e il bivio con l&#39;intestino in metamorfico lampreda di mare. Ilprocesso di degenerazione biliare è stato visualizzato e discernimento durante la lampreda di mare metamorfosi mediante scansione laser microscopia confocale. Questo metodo fornisce un potente strumento per studiare atresia biliare in un modello animale unico.

Diversi importanti eventi evolutivi si verificano nel sistema epatobiliare durante la lampreda di mare metamorfosi. Il dotto biliare e la cistifellea apoptosi e degenerata (Figura 1). Combinando il metodo chiarimento modificato e colorazione con fegato marker delle cellule citocheratina 19 (CK19, presente in entrambi i colangiociti ed epatociti prima e dopo la metamorfosi 13) e anti-apoptotica Bcl2 marcatore utilizzando la microscopia confocale, l&#39;intero sistema biliare è stato catturato...

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

Lampreda di mare perde la cistifellea e vie biliari durante la metamorfosi, un processo simile a atresia biliare umano. Un nuovo metodo di fissazione e chiarificazione (CLARITY) è stato modificato per visualizzare l&#39;intero albero biliare mediante scansione laser microscopia confocale. Questo metodo fornisce un potente strumento per studiare la degenerazione biliare.

Un metodo Chiarimento Nuovo per visualizzare biliare degenerazione Durante fegato Metamorfosi in mare Lamprey ( 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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JoVE Journal

Biology

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

13.7K Views.  Michigan State University.  Lampreda di mare perde la cistifellea e vie biliari durante la metamorfosi, un processo simile a atresia biliare umano. Un nuovo metodo di fissazione e chiarificazione (CLARITY) è stato modificato per visualizzare l'intero albero biliare mediante scansione laser microscopia confocale. Questo metodo fornisce un potente strumento per studiare la degenerazione biliare. 

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.

Pirosequenziamento: un metodo semplice per genotipizzazione accurata

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

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Biologia

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.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

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.

L&#39;uso della microscopia Time lapse di visualizzare anossia indotta Suspended Animation in<em&gt; C. elegans</em&gt; Embrioni

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

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.

High Throughput microiniezioni di Sea Urchin zigoti

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.

Un metodo per estrarre pigmenti da Squid<em&gt; Pealeii Doryteuthis</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.

Utilizzando un metodo immunohistochemico integrale per studiare l&#39;innervazione del tratto bipolare in<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.

Microdilution del brodo In Vitro Screening: un metodo facile e veloce per rilevare nuovi composti Antifungini

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.

Proteina fluorescente verde sistema di visualizzare gli effettori consegnati dai batteri durante l'infezione Split

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 ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

Immunolabelling Degenerazione di mitragliari nelle biopsie muscolari

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

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.

Induzione e rilevamento dell'apoptosi in una cultura primaria delle cellule intestinali del cetriolo marino

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