Name: rna in situ hybridization in whole mount embryos and cell histology adapted for marine elasmobranchs
Uploaded: 2013-04-12T12:00:00
Description: Watch this Scientific Journal Video about RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs at JoVE.com

Jeg vil gerne takke de mange studerende, der har arbejdet i mit laboratorium og bidrog til udviklingen af ​​disse protokoller. NAT har modtaget støtte fra Skidmore-Union Network, et projekt etableret med en NSF forskud tilskud.

I. RNA Whole Mount in situ hybridisering i Marine bruskfisk 1. Embryo Fiksering og klargøring <ol><li> Skate embryoner kan iscenesættes i henhold til Ballard 7. Optimale trin til påvisning af genekspression afhænger af vævet af interesse. Sådan sporer genekspression i skate fordøjelseskanalen, stadier 27-30 er optimale 7. </li><li> Dissekere embryoner i PBS, adskillelse af embryoner fra blommesækken. </li><li> Fix i 30 ml frisk fremstillet 4% paraformald...

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L., Laufer, E., Tabin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+mediates+the+polarizing+activity+of+the+ZPA.">Sonic hedgehog mediates the polarizing activity of the ZPA.</a> Cell. 75, 1401-1416 (1993).</li><li>Theodosiou, N. A., Hall, D. A., Jowdry, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+acid+mucin+goblet+cell+distribution+and+Hox13+expression+patterns+in+the+developing+vertebrate+digestive+tract.">Comparison of acid mucin goblet cell distribution and Hox13 expression patterns in the developing vertebrate digestive tract.</a> J. Exp. Zool. B. Mol. Dev. Evol. 308, 442-453 (2007).</li><li>Sambrook, J., Russell, D. 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De præsenterede protokoller er klassiske metoder til overvågning af genekspression og identifikation af differentierede celletyper, og er indrettet til brug i marine bruskfisk. Yderligere modifikationer af disse protokoller kan det være nødvendigt at tilpasse sig forskellige selachier. Den mest almindelige bekymring RNA hele mount in situ s er risikoen for RNase kontaminering og dermed nedbrydning af RNA-proben og endogene meddelelser. To aspekter skal tages i betragtning: synte...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>10 x transcription buffer</td>
 <td>Roche</td>
 <td>11-465-384-001</td>
 <td></td>
 </tr>
 <tr>
 <td>DIG-RNA labeling mix</td>
 <td>Roche</td>
 <td>11-277-073-910</td>
 <td></td>
 </tr>
 <tr>
 <td>RNAse inhibitor</td>
 <td>Roche</td>
 <td>03-335-399-001</td>
 <td></td>
 </tr>
 <tr>
 <td>RNA polymerase - SP6</td>
 <td>Roche</td>
 <td>10-810-274-001</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAseI, RNAse-free </td>
 <td>Roche</td>
 <td>10-776-785-001</td>
 <td></td>
 </tr>
 <tr>
 <td>Yeast RNA</td>
 <td>Invitrogen</td>
 <td>15401-029</td>
 <td></td>
 </tr>
 <tr>
 <td>CHAPS</td>
 <td>EMD-Millipore</td>
 <td>220201</td>
 <td></td>
 </tr>
 <tr>
 <td>heparin</td>
 <td>Sigma-Aldrich</td>
 <td>H4784</td>
 <td></td>
 </tr>
 <tr>
 <td>DEPC (diethyl pyrocarbonate)</td>
 <td>Research Organics</td>
 <td>2106D</td>
 <td></td>
 </tr>
 <tr>
 <td>Moria Perforated Spoon</td>
 <td>Fine Science Tools</td>
 <td>10370-17</td>
 <td></td>
 </tr>
 <tr>
 <td>Netwell inserts</td>
 <td>Electron Microscopy Sciences</td>
 <td>64713-00</td>
 <td>Netwells for use in 6-well tissue culture dishes</td>
 </tr>
 <tr>
 <td>6-well tissue culture plate</td>
 <td>Corning</td>
 <td>3516</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass scintillation vials with screw-cap lids</td>
 <td>Weaton Science Products</td>
 <td>986540</td>
 <td></td>
 </tr>
 <tr>
 <td>formamide</td>
 <td>Fisher</td>
 <td>BP227500</td>
 <td></td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>Invitrogen</td>
 <td>59895 (AM2542)</td>
 <td></td>
 </tr>
 <tr>
 <td>NBT</td>
 <td></td>
 <td>11585029001</td>
 <td></td>
 </tr>
 <tr>
 <td>BCIP</td>
 <td>Roche</td>
 <td>11585002001</td>
 <td></td>
 </tr>
 <tr>
 <td>Hydrogen peroxide, 30%</td>
 <td>EMD</td>
 <td>HX0635-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Sheep serum</td>
 <td>VWR</td>
 <td>101301-478</td>
 <td></td>
 </tr>
 <tr>
 <td>glutaraldehyde</td>
 <td>Sigma-Aldrich</td>
 <td>G5882</td>
 <td></td>
 </tr>
 <tr>
 <td>tRNA</td>
 <td>Roche</td>
 <td>10-109-541-001</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-DIG Fab Fragments</td>
 <td>Roche</td>
 <td>1137-6623</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 3. Reagents and equipment for RNA whole mount in situ's.</td>
 </tr>
 <tr>
 <td>1% Alcian Blue 8GS, pH 2.5</td>
 <td>Electron Microscopy Sciences</td>
 <td>26323-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Nuclear Fast Red</td>
 <td>Electron Microscopy Sciences</td>
 <td>26078-05</td>
 <td></td>
 </tr>
 <tr>
 <td>DPX Mountant</td>
 <td>Electron Microscopy Sciences</td>
 <td>13510</td>
 <td></td>
 </tr>
 <tr>
 <td>Paraffin (Paraplast X-tra)</td>
 <td>McCormick Scientific</td>
 <td>39503002</td>
 <td></td>
 </tr>
 <tr>
 <td>10% Formalin, NBF</td>
 <td>VWR</td>
 <td>95042-908</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass scintillation vials with screw-cap lids</td>
 <td>Weaton Science Products</td>
 <td>986540</td>
 <td></td>
 </tr>
 <tr>
 <td>Stainless steal base molds</td>
 <td>Tissue-Tek</td>
 <td>4161-4165</td>
 <td>Multiple sizes available.</td>
 </tr>
 <tr>
 <td>Cassettes </td>
 <td>Tissue-Tek</td>
 <td>4170</td>
 <td></td>
 </tr>
 <tr>
 <td>Slide warmer</td>
 <td>Fisher-Scientific</td>
 <td>12-594Q</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue Embedder</td>
 <td>Leica Microsystems</td>
 <td>EG1160</td>
 <td></td>
 </tr>
 <tr>
 <td>Microtome, rotary</td>
 <td>Leica Microsystems</td>
 <td>RM2235</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue-Tek Slide Staining Set</td>
 <td>Electron Microscopy Sciences</td>
 <td>62540-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue-Tek 24-Slide Holder</td>
 <td>Electron Microscopy Sciences</td>
 <td>62543-06</td>
 <td></td>
 </tr>
 <tr>
 <td>Superfrost*Plus slides</td>
 <td>Fisherbrand</td>
 <td>12-550-17</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 4. Reagents and equipment for Alcian Blue stain.</td>
 </tr>
 </tbody>
</table>

rna in situ hybridization in whole mount embryos and cell histology adapted for marine elasmobranchs

Marine bruskfisk værdiansættes dyremodeller for biomedicinske og genomiske studier, da de er de mest primitive hvirveldyr, der har adaptive immunitet og har unikke mekanismer til osmoregulering 1-3. Som de mest primitive levende stålkæber-hvirveldyr med parrede vedhæng, er bruskfisk en evolutionært vigtig model, især for studier i evolution og udvikling. Marine bruskfisk er også blevet brugt til at studere akvatisk toksikologi og stress fysiologi i relation til klimaændringer 4. Således er udvikling og tilpasning af metoder er nødvendige for at lette og udvide brugen af ​​disse primitive hvirveldyr til flere biologiske discipliner. Her vil jeg præsentere en vellykket tilpasning af RNA hele mount in situ hybridisering og histologiske teknikker til at studere genekspression og celle histologi i bruskfisk. Overvågning genekspression er kendetegnende værktøj af udviklingstoksicitet biologists, og er i vid udstrækning anvendes til at undersøge udviklingsmæssige processer 5. RNA hele mount in situ hybridisering muliggør visualisering og lokalisering af specifikke gen-transkripter i væv i det udviklende embryo. Udtrykket mønster af et gen budskab kan give indblik i, hvad udviklingsprocesser og celle skæbne beslutninger et gen kan styre. Ved at sammenligne ekspressionsmønsteret af et gen på forskellige udviklingstrin, kan opnås indsigt i, hvordan den rolle, et gen ændres under udviklingen. Medens hele mount in situ &#39;s tilvejebringer et middel til at lokalisere genekspression til væv, histologiske teknikker muliggøre identifikation af differentierede celletyper og væv. Histologiske pletter har mangfoldige funktioner. Generelle pletter benyttes til at fremhæve cellemorfologi, fx hematoxylin og eosin til generel farvning af kerner og cytoplasma, hhv. Andre pletter kan fremhæve bestemt celletyper. For eksempel plette Alcian blå rapporteret i dette papir er et meget anvendt kationisk pletten til at identificere mucosaccharides. Farvning af fordøjelseskanalen med Alcian Blue kan identificere fordelingen af ​​slimceller, der producerer mucosaccharides. Variationer i mucosaccharide bestanddele på korte peptider skelne slimceller efter funktion som fordøjelseskanalen 6. Ved at bruge RNA hele mount in situ &#39;s og histokemiske metoder samtidigt, kan celle skæbne beslutninger knyttet til gen-ekspression. Selv RNA in situ &#39;s og histokemi er meget brugt af forskere, har deres tilpasning og brug i marine bruskfisk mødt begrænset og varieret succes. Her vil jeg præsentere protokoller udviklet for bruskfisk og bruges på en regelmæssig basis i mit laboratorium. Selv om en yderligere modifikation af RNA in situ &#39;s hybridiseringsmetode kan være nødvendigt at tilpasse sig forskellige arter, de protokoller, der beskrives her provide et stærkt udgangspunkt for forskere, der ønsker at tilpasse anvendelsen af ​​marine bruskfisk til deres videnskabelige undersøgelser.

Eksempler på Alcian Blue farvning i forskellige regioner af L. erinacea fordøjelseskanalen er vist i figur 2. Acid mucin indeholdende globlet celler er klart synlige ved Alcian Blue farvning i hele fordøjelseskanalen. Fordelingen af ​​sure muciner forskellig i de forskellige regioner i fordøjelseskanalen, hvilket afspejler forskelle i funktion. Sure muciner er tyndt produceres i spiral tarmen og cloaca, medens en høj koncentration af sure muciner påvises i den distale tarmen (sammenli...

Watch this Scientific Journal Video about RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs at JoVE.com

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Ved at kombinere metoder til RNA hele mount<em&gt; In situ</em&gt; Hybridisering og histologi, kan genekspression være forbundet med celle skæbne beslutninger i det udviklende foster. Disse metoder er blevet tilpasset til marine bruskfisk og lette brugen af ​​disse dyr som modelorganismer til biomedicinsk, toksikologi og sammenlignende undersøgelser.

RNA<em&gt; In situ</em&gt; Hybridisering i Hele Mount Embryoner og Cell Histologi Tilpasset til Marine bruskfisk

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

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Biology

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

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This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...