Name: Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues (Video) | JoVE
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Denne undersøgelse blev støttet af tilskud fra National Natural Science Foundation of China (82204951) og Sichuan Science and Technology Program (2020JDRC0102).

Voksne C57BL/6-mus (6 uger, 25-30 g) blev anvendt i dette studie. Alle procedurer blev udført i overensstemmelse med lokale regler for dyrevelfærd og eksperimentel etik. Undersøgelsen blev godkendt af West China Hospital of Sichuan University Biomedical Research Ethics Committee.
1. Glomeruli mærkning og vævsforberedelse
<ol><li>Glomeruli mærkning<ol><li>FITC-dextran (10 mg) opløses i 1x fosfatbufret saltvand (PBS) i forholdet 1:1 (1 mg: 1 ml) for at forberede arbejds...

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

<ol>
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G., 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+number+and+size+variability+and+risk+for+kidney+disease.">Glomerular number and size variability and risk for kidney disease.</a> Current Opinion in Nephrology and Hypertension. 20 (1), 7-15 (2011).</li><li>Bertram, J. F., 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=Why+and+how+we+determine+nephron+number.">Why and how we determine nephron number.</a> Pediatric Nephrology. 29, 575-580 (2014).</li><li>Bertram, J. F., Soosaipillai, M. C., Ricardo, S. D., Ryan, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+numbers+of+glomeruli+and+individual+glomerular+cell+types+in+the+normal+rat+kidney.">Total numbers of glomeruli and individual glomerular cell types in the normal rat kidney.</a> Cell and Tissue Research. 270 (1), 37-45 (1992).</li><li>Nyengaard, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereologic+methods+and+their+application+in+kidney+research.">Stereologic methods and their application in kidney research.</a> Journal of the American Society of Nephrology. 10 (5), 1100-1123 (1999).</li><li>Bertram, J. 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G., Moeller, M. J., Bertram, J. 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Vævsrensningsteknologier kan klassificeres i 3 eller 4 grupper 29,30,31. Organisk opløsningsmiddelbaseret vævsrensning (f.eks. DISCO og PEGASOS), vandig vævsrensning (f.eks. CUBIC) og hydrogelindlejringsvævsrensning (f.eks. CLARITY) er alle blevet anvendt til nyrerensning 25,26,28,32. CUBIC, som v...

Antallet og mængden af glomeruli er meget vigtigt for diagnosticering og behandling af forskellige nyresygdomme 1,2,3,4,5. Den gyldne standard for glomeruli nummer estimering er den fysiske dissektor / fraktionator kombination. Denne metode kræver dog specielle reagenser og udstyr, hvilket gør den langsom og dyr 6,7,8,9. Biopsi giver et væld af oplysninger, men denne metode er naturligvis kun egnet til grove estimater10,11. Medicinske billeddannelsesteknologier, herunder magnetisk resonansbilleddannelse (MR), computertomografi (CT) og røntgen, anvendes også i vid udstrækning i glomerulær detektion 12,13,14,15, men sådanne teknologier kræver omfangsrige instrumenter. Nye metoder, såsom matrixassisteret laserdesorption / ionisering (MALDI) billeddannelsemassespektrometer 16 eller tyk og tynd sektion metode17, er også blevet brugt til glomerulær detektion, selvom de forbliver kedelige og besværlige.
Ved hjælp af gennemsigtighedsteknologier er det muligt at observere dybere dybder og opnå rigere og mere komplet information fra tykt væv eller endda hele organer 18,19,20,21,22,23. Derfor er gennemsigtighedsteknologier blevet anvendt i vid udstrækning i nyreforskning24. Observation og påvisning af glomeruli i de ryddede nyrer er også involveret. Imidlertid henviste disse offentliggjorte artikler enten kun kort til glomerulær detektion25 eller brugte vanskelige at opnå mærkningsmetoder såsom transgene dyr26, selvproducerede farvestoffer13 eller antistofinkubation med høj koncentration27 til mærkning af glomeruli. Hertil kommer, at selv om undersøgelser havde analyseret glomeruli i ryddede nyrer, var analyserne altid begrænsede13 eller baseret på analysealgoritmer etableret af forfatterne selv26.
Vi har tidligere demonstreret en mere bekvem måde at mærke glomeruli hos mus nyrer28. Ved at bruge Imaris fandt vi, at glomerulital, frekvens og volumen hurtigt kunne opnås. Således præsenterer vi her et mere tilgængeligt, omfattende og forenklet sæt metoder til mærkning og analyse af glomeruli hos mus nyrer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% PFA</td>
			<td>Biosharp</td>
			<td>7007171800</td>
			<td>Fixation reaagen</td>
		</tr>
		<tr>
			<td>502 Glue&#160;</td>
			<td>Deli</td>
			<td>7146</td>
			<td>For fixing the kidney to the sample fixing adapter&#160;</td>
		</tr>
		<tr>
			<td>Antipyrine</td>
			<td>Aladdin</td>
			<td>A110660</td>
			<td>Clearing reagent</td>
		</tr>
		<tr>
			<td>Brain Matrix</td>
			<td>RWD Life Science</td>
			<td>1mm 40-75</td>
			<td>Tissue slicing</td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Nikon</td>
			<td>A1plus</td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>FITC-Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD150S</td>
			<td>Labeling reagent</td>
		</tr>
		<tr>
			<td>Light sheet fluorescence microscopy&#160;</td>
			<td>Zeiss</td>
			<td>Light sheet 7&#160;</td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Ensiweier</td>
			<td></td>
			<td>Adult C57BL/6 mice (6 weeks of age, 25&#8211;30 g)&#160;</td>
		</tr>
		<tr>
			<td>N-Butyldiethanolamine</td>
			<td>Aladdin</td>
			<td>B299095</td>
			<td>Clearing reagent</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Aladdin</td>
			<td>N105042</td>
			<td>Clearing reagent</td>
		</tr>
		<tr>
			<td>Pentobarbital Natriumsalz</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr>
			<td>Tail vein fixator</td>
			<td>JINUOTAI</td>
			<td>JNT-FS35</td>
			<td>Fix the mouse for vail injection</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Clearing reagent</td>
		</tr>
	</tbody>
</table>

an efficient and fast method for labeling and analyzing mouse glomeruli

Glomeruli er grundlæggende enheder i nyrerne; Derfor er undersøgelse af glomeruli afgørende for forståelsen af nyrefunktion og patologi. Biologisk billeddannelse giver intuitiv information; Det er således af stor betydning at mærke og observere glomeruli. Imidlertid kræver glomeruli-observationsmetoderne, der i øjeblikket er i brug, komplicerede operationer, og resultaterne kan miste etiketdetaljer eller tredimensionel (3D) information. Den klare, uhindrede hjernebilleddannelsescocktails og beregningsanalyse (CUBIC) vævsrensningsteknologi er blevet brugt i vid udstrækning i nyreforskning, hvilket giver mulighed for mere nøjagtig detektion og dybere detektionsdybde. Vi fandt ud af, at museglomeruli hurtigt og effektivt kan mærkes ved haleveneinjektion af FITC-Dextran med medium molekylvægt efterfulgt af CUBIC clearing-metoden. Den ryddede musenyre kunne scannes af et lysarkmikroskop (eller et konfokalmikroskop, når det skæres) for at opnå tredimensionelle billedstakke af alle glomeruli i hele nyrerne. Behandlet med passende software kunne glomeruli-signalerne let digitaliseres og analyseres yderligere for at måle antallet, volumenet og frekvensen af glomeruli.

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

Denne undersøgelse giver en enkel og effektiv metode til mærkning og analyse af glomeruli hos mus nyrer.
Glomeruli (blodkar) kan mærkes godt ved intravaskulært injiceret FITC-Dextran. Efter clearingprocessen blev nyrerne gennemsigtige (figur 1A), og glomeruli kunne tydeligt observeres ved hjælp af lysarkmikroskopi (figur 1B) eller konfokalmikroskopi (figur 1C). Konfokal mikroskopi har en begrænset s...

Watch this Scientific Journal Video about Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues at JoVE.com

Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues (Video) | JoVE

Denne undersøgelse præsenterer et brugervenligt, komplet og simpelt sæt metoder til at mærke og analysere glomeruli fra CUBIC-ryddede musenyrer. Data såsom glomerulus antal og volumen kan opnås let og pålideligt ved hjælp af fluorescein isothiocyanat (FITC)-Dextran, lysark fluorescens mikroskopi (LSFM), eller fælles konfokal mikroskopi og software såsom Imaris.

En effektiv og hurtig metode til mærkning og analyse af museglomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...

Mærkning og tælling af glomeruli ved hjælp af konventionelle metoder kan være kompliceret, kedeligt, tidskrævende og omfangsrigt instrument er påkrævet. Vores metode tilbyder en øjeblikkelig og intuitivt nøjagtig mærkningsmetode til analyse af glomeruli i hele nyrerne. Vores teknik er en hurtig, nem, præcis og billig metode til at analysere glomeruli i hele nyren.Det kræver ikke noget specielt udstyr, så det kan udføres i ethvert laboratorium. Dette er en banebrydende, hurtig og enkel metode til at mærke glomeruli, hvilket giver mulighed for en intuitiv observation af alle glomeruli i hele nyrerne. Det har stort potentiale til at hjælpe forskere med at gøre betydelige gennembrud i undersøgelsen af nyrerne ved at give information såsom glomeruli mængde, volumen, distribution og mere.På den ene side undersøger vi anvendelsen af denne glomeruli-mærkningsmetode i klinisk nyresygdomsforskning. På den anden side vil vi fortsætte med at arbejde på at forbedre metoder til at mærke biotargets som blodkar eller specifikke proteiner i gennemsigtigt væv og justere dem for at imødekomme vores forskningsbehov.