Name: quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in munich wistar rats
Uploaded: 2013-04-17T12:00:00
Description: Watch this Scientific Journal Video about Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats at JoVE.com

Forfatterne vil gerne takke Drs Silvia B Campos-Bilderback og George J Rhodes til at udfylde kirurgiske procedurer, der involverer placering af venøse adgang linjer. De vil også gerne takke Sara E Wean for at opretholde München-Wistar kolonier bestående af både Simonsen og Frömter stammer.

1. Konjugering af rotteserumalbumin til Sulfo-rhodamin 101 sulfonylchlorid (Texas Red) <ol><li> Opløs 100 mg rotteserumalbumin (RSA) i 6.667 ml 100 mM natriumbikarbonat pH 9,0, slutkoncentration 15 mg / ml i en 50 ml konisk rør. </li><li> Placer løsning i is / vand bægerglas og afkøles til mellem 0 og 4 ° C. </li><li> Tilsæt 200 pi af høj kvalitet vandfrit dimethylformamid (DMF) til en 10 mg hætteglas af Texas Red sulfonylchlorid (TRSCs), vortex på medium i 15 sek. </li><li> Vortex RSA løsning på medium og...

<ol>
	<li>Russo, L. 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=The+normal+kidney+filters+nephritic+levels+of+albumin+retrieved+by+proximal+tubule+cells;+retrieval+is+disrupted+in+nephritic+states.">The normal kidney filters nephritic levels of albumin retrieved by proximal tubule cells; retrieval is disrupted in nephritic states.</a> Kidney International. 71, 504 (2007).</li><li>Russo, L. 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=Impaired+tubular+uptake+explains+albuminuria+in+early+diabetic+nephropathy.">Impaired tubular uptake explains albuminuria in early diabetic nephropathy.</a> Journal of the American Society of Nephrology. 20 (3), 489 (2009).</li><li>Sandoval, R. 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=Multiple+factors+influence+glomerular+albumin+permeability+in+rats.">Multiple factors influence glomerular albumin permeability in rats.</a> Journal of the American Society of Nephrology. 23 (3), 447 (2012).</li><li>Tojo, A., Endou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrarenal+handling+of+proteins+in+rats+using+fractional+micropuncture+technique.">Intrarenal handling of proteins in rats using fractional micropuncture technique.</a> American Journal of Physiology. 263, 601 (1992).</li><li>Asgeirsson, 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=Glomerular+sieving+of+three+neutral+polysaccharides,+polyethylene+oxide+and+bikunin+in+rat:+Effects+of+molecular+size+and+conformation.">Glomerular sieving of three neutral polysaccharides, polyethylene oxide and bikunin in rat: Effects of molecular size and conformation.</a> Acta Physiologica. 191 (3), 237 (2007).</li><li>Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+endocytosis+in+vivo+using+intravital+two-photon+microscopy.">Quantifying endocytosis in vivo using intravital two-photon microscopy.</a> Methods in Molecular Biology. 440, 389 (2008).</li><li>Dunn, K. W., 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=Live-animal+imaging+of+renal+function+by+multi-photon+microscopy.">Live-animal imaging of renal function by multi-photon microscopy.</a> Curr. Protoc. Cytom. Chapter 12, Unit 12.9 (2007).</li></ol>

Trinene fremhævet her repræsenterer, hvad vi føler for at være dem, der vil producere konsistente og præcise permeabilitetsværdier fordi de omgå de følgende faldgruber: <ol><li> Spredning: Brugen af ​​en rød udsender fluorophores give mulighed for mere effektiv indsamling af lys, da længere bølgelængde fotoner er mindre tilbøjelige til at sprede. Brug enten grønne eller blå udsender fluoroforer vil indføre større variation i generalsekretariatets på grund af den øgede variation i intensitet v?...

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 <td>Olympus Floview 1000 confocal/Multi-photon microscope</td>
 <td>Olympus America</td>
 <td></td>
 <td>Filters for detectors: Blue 430/100, Green 525/50, Red 605/90</td>
 </tr>
 <tr>
 <td>Mode-Locked Ti:Sapphire Mai Tai Laser</td>
 <td>Spectra-Physics</td>
 <td></td>
 <td>Tunable excitation wavelengths: ~750-1150 nm</td>
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 <td>Gallium arsenide phosphide photodetectors</td>
 <td>Hamamatsu Corp</td>
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 <td>Note: Front or Side mounted configurations available.</td>
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 <td>Metamorph Image processing Software</td>
 <td>Molecular Dynamics</td>
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 <td>Note: Version 6.1r1</td>
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 <td>Microsoft Excel</td>
 <td>Microsoft Corportation</td>
 <td></td>
 <td>2007 version</td>
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 <td>Handling Forceps</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 78266-04</td>
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 <td>Mayo Dissecting Scissors</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 72962</td>
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 <td>CA Micro scissors Model 1C300</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 78180-1C3</td>
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 <td>Kelly Hemostatic Forceps (straight)</td>
 <td>Electron Microscopy Sciences</td>
 <td>Cat# 72930</td>
 <td></td>
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 <td>Water Jacket Blanket + Heating Pad</td>
 <td>Gaymar</td>
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 <td>T/Pump PN 11184-000 Blanket-66N111CC</td>
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 <td>Repti-Therm Under Tank Heater</td>
 <td>ZooMed</td>
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 <td>RH-4</td>
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 <td>Texas Red Sulfonyl Chloride</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>Cat# T-353</td>
 <td></td>
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 <td>Rat Serum Albumin</td>
 <td>Sigma Aldrich</td>
 <td>Cat# A-6272</td>
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 <td>High Quality Anhydrous DMF</td>
 <td>Sigma Aldrich</td>
 <td>Cat# 270547</td>
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 <td>Strate-Line Autoclave Tape</td>
 <td>Fisher Scientific</td>
 <td>Cat# 11-889-1</td>
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 <td>Willco-dish Coverslip Bottom Dishes (50 mm/40 mm coverslip)</td>
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</table>

quantifying glomerular permeability of fluorescent macromolecules using 2-photon microscopy in munich wistar rats

Nyresygdomme involverer urin tab af store væsentlige makromolekyler, såsom serumalbumin, har længe været anset for at være forårsaget af ændringer i permeabiliteten barriere består af podocytes, vaskulære endotelceller, og en basalmembran arbejder i fællesskab. Data fra vores laboratorium ved hjælp intravital 2-foton mikroskopi afslørede en mere permeabel glomerulær filtration barriere (GFB) end tidligere antaget under fysiologiske betingelser, med genfinding af filtreret albumin forekommer i en tidlig delmængde af celler, der kaldes proximale tubuliceller (PTC) 1,2, 3.. Tidligere teknikker, der anvendes til at studere renal filtrering og oprettelse af karakteristisk for filtrering barriere involverede micropuncture af lumen af disse tidlige rørformede segmenter med prøveudtagning af væske indhold og analyse 4.. Disse undersøgelser fastlagt albumin koncentrationen i den luminale væske til at være næsten ikke-eksisterende, svarende nøjehvilket normalt påvises i urinen. Imidlertid karakterisering af dextran polymerer med definerede størrelser af denne teknik viste de en størrelse svarende til serumalbumin havde højere niveauer i den rørformede lumen og urin, hvilket tyder øget gennemtrængelighed 5.. Heri er en detaljeret skitse af teknik, der anvendes til direkte at visualisere og kvantificere glomerulær fluorescerende albumin permeabilitet in vivo. Denne metode giver mulighed for påvisning af filtreret albumin tværs filtrering barrieren til Bowmans rum (den oprindelige kammer urin filtrering), og også tillader kvantificering af albumin reabsorption af proximale tubuli og visualisering af efterfølgende albumin transcytose 6.. Fraværet af fluorescerende albumin langs senere rørformede segmenter undervejs til blæren fremhæver effektiviteten af ​​hentning vej i de tidligere proksimale segmenter. Hertil kommer, at når denne teknik blev anvendt bestemme permeabilitetenaf dextraner med en lignende størrelse til albumin næsten ens permeabilitetsværdier blev rapporteret 2. Disse observationer er direkte støtter behovet for at udvide fokus for mange proteinuric nyresygdomme til omfattede ændringer i proksimal tubulus celle regenerering.

Figur 3 viser et eksempel på et billede taget fra en overflade glomerulus en München Wistar Frömter rotter og de ​​skridt til at bestemme permeabiliteten af fluorescerende albumin. GSC værdi for albumin af 0,0111 udledt for denne person glomerulus falder inden for intervallet ses i denne stamme af München Wistarrotter når i fastende tilstand 3.. Stabiliteten set i disse billeder skyldes omhyggelig planlægning og udførelse af instruktioner afbildet i figur 1 og 2. ...

Watch this Scientific Journal Video about Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats at JoVE.com

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

En teknik udnytter høj opløsning intavital 2-foton mikroskopi til direkte at visualisere og kvantificere gloemrular filtrering i overfladen glomeruli. Denne metode giver mulighed for direkte bestemmelse af permeabilitetsegenskaber af makromolekyler i både normale og syge tilstande.

Kvantificering Glomerular fyldbarhed Fluorescent Macromolecules Brug 2-Photon mikroskopi i München Wistarrotter

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

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