Name: Identificatie van de bron van uitgescheiden eiwitten in de nier door Brefeldin A-injectie (Video) | JoVE Journal
Uploaded: 2021-11-10T13:00:00.000+00:00
Duration: 10 min 15 s
Description: Leer een methode met Brefeldin A-injectie om cytokine-producerende cellen in beschadigde nieren te identificeren. Dit protocol maakt kwalitatieve analyse van eiwitten in nierepitheel- en interstitiële cellen mogelijk.

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5 minutes

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</ol>

<table><tbody><tr><td>1 mL Insulin syringes</td><td>BD</td><td>329654</td><td></td></tr><tr><td>10 mL Syringe</td><td>BD</td><td>302995</td><td></td></tr><tr><td>2 mL tube</td><td>Fisher brand</td><td>05-408-138</td><td></td></tr><tr><td>20 mL Syringe</td><td>BD</td><td>302830</td><td></td></tr><tr><td>25 G needles</td><td>BD</td><td>305125</td><td></td></tr><tr><td>28 G needles</td><td>BD</td><td>329424</td><td></td></tr><tr><td>96-well-plate</td><td>Corning</td><td>9017</td><td></td></tr><tr><td>Aristolochic acid-I</td><td>Sigma-Aldrich</td><td>A9461</td><td></td></tr><tr><td>&alpha;-SMA antibody conjugated with Cy3</td><td>Sigma-Aldrich</td><td>C6198</td><td>RRID:AB_476856</td></tr><tr><td>Blade for cryostat</td><td>C.L. Sturkey. Inc</td><td>DT315R50</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>&nbsp;Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma-Aldrich</td><td>B6542</td><td></td></tr><tr><td>Cisplatin</td><td>Sigma-Aldrich</td><td>P4394</td><td></td></tr><tr><td>Citric acid</td><td>Sigma-Aldrich</td><td>791725</td><td></td></tr><tr><td>Confocal microscope</td><td>ZEISS</td><td>&nbsp;LSM710</td><td></td></tr><tr><td>Confocal microscopy objectives</td><td>ZEISS</td><td>40x / 1.10 LD C-Apochromat WATER</td><td></td></tr><tr><td>Confocal software</td><td>ZEISS</td><td>ZEN</td><td></td></tr><tr><td>Coplin jar</td><td>Fisher Scientific</td><td>19-4</td><td></td></tr><tr><td>Cover glass</td><td>Fisher brand</td><td>12545F</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1850</td><td></td></tr><tr><td>CTGF antibody</td><td>Genetex</td><td>GTX124232</td><td>RRID:AB_11169640</td></tr><tr><td>Cy3-AffiniPure Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson immunoresearch</td><td>711-165-152</td><td>RRID: AB_2307443</td></tr><tr><td>Cy5-AffiniPure Donkey Anti-Goat IgG (H+L)</td><td>Jackson immunoresearch</td><td>705-175-147</td><td>RRID: AB_2340415</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Disposable base molds</td><td>Fisher brand</td><td>22-363-553</td><td></td></tr><tr><td>donkey serum</td><td>Jackson immunoresearch</td><td>017-000-121</td><td></td></tr><tr><td>Ethanol</td><td>Decon Labs, Inc.</td><td>2701</td><td></td></tr><tr><td>Forceps</td><td>VETUS</td><td>ESD-13</td><td></td></tr><tr><td>Glycine</td><td>Fisher brand</td><td>12007-0050</td><td></td></tr><tr><td>Heating pads</td><td>Kent scientific</td><td>DCT-20</td><td></td></tr><tr><td>Heparin sodium salt</td><td>ACROS organics</td><td>41121-0010</td><td>100mg/15ml of dH2O</td></tr><tr><td>Humidified chamber</td><td>Invitrogen</td><td>44040410</td><td>A plastic box covered in foil can be used as an alternative humidified chamber.</td></tr><tr><td>Insulin syringes</td><td>BD</td><td>329461</td><td></td></tr><tr><td>Inverted microscope</td><td>NIKON</td><td>Eclipse Ti-E2</td><td>immunofluorescence</td></tr><tr><td>KIM-1 antibody</td><td>R &amp; D</td><td>AF1817</td><td>RRID: AB_2116446</td></tr><tr><td>Lemozole (Histo-clear)</td><td>National diagnostics</td><td>HS-200</td><td></td></tr><tr><td>lotus tetragonolobus lectin</td><td>Vector</td><td>FL-13212</td><td></td></tr><tr><td>Microscope slide</td><td>Fisher scientific</td><td>12-550-343</td><td></td></tr><tr><td>Microtome</td><td>Reichert</td><td>Jung 820 II</td><td></td></tr><tr><td>monochrome CMOS camera</td><td>NIKON</td><td>&nbsp;DS-Qi-2</td><td></td></tr><tr><td>Mouse surgical kit</td><td>Kent scientific</td><td>INSMOUSEKIT</td><td></td></tr><tr><td>NIS Elements</td><td>NIKON</td><td></td><td></td></tr><tr><td>Objectives</td><td>NIKON</td><td>Plan Apo 20x/0.75</td><td>image acquisition software linked to Eclipse Ti-E2 (invertd microscope)</td></tr><tr><td>OCT compound</td><td>Scigen</td><td>4586</td><td></td></tr><tr><td>Pap pen</td><td>Vector</td><td>H-4000</td><td></td></tr><tr><td>PBS with calcium and magnesium</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>PBS without calcium and magnesium</td><td>&nbsp;Corning</td><td>21-031-CV</td><td></td></tr><tr><td>PDGF-D antibody</td><td>Thermo-Fisher scientific</td><td>40-2100</td><td></td></tr><tr><td>PFA</td><td>Electron Microscopy Science</td><td>15710</td><td>RRID: AB_2533455</td></tr><tr><td>Plate reader</td><td>Promega</td><td>GloMax&reg; Discover Microplate Reader</td><td>4% PFA is diluted from 16% in PBS.</td></tr><tr><td>povidone-iodine (Betadine)</td><td>Avrio Health L.P.</td><td>NDC 67618-151-17</td><td></td></tr><tr><td>Pressure cooker</td><td>&nbsp;Tristar 8</td><td>Qt. Power Cooker Plus</td><td></td></tr><tr><td>ProLong Gold Antifade Reagent</td><td>Invitrogen</td><td>P36930</td><td></td></tr><tr><td>Quantichrom Urea (BUN) assay Kit II</td><td>BioAssay Systems</td><td>DUR2-100</td><td></td></tr><tr><td>Single-edge razor blade for kidney dissection (.009", 0.23 mm)</td><td>IDL tools</td><td>521013</td><td></td></tr><tr><td>Slide warmer</td><td>Lab Scientific Inc.,</td><td>XH-2001</td><td></td></tr><tr><td>Software</td><td>NIKON</td><td>NIS elements</td><td></td></tr><tr><td>Sucrose</td><td>RPI</td><td>S24060</td><td></td></tr><tr><td>TGF-b1 anitbody</td><td>Sigma-Aldrich</td><td>SAB4502954</td><td></td></tr><tr><td>Tris EDTA buffer</td><td>Corning</td><td>46-009-CM</td><td>RRID: AB_10747473</td></tr><tr><td>Trisodium citrate dihydrate</td><td>Sigma-Aldrich</td><td>SLBR6660V</td><td></td></tr><tr><td>Trisodium citrate dihydrate</td><td>Sigma-Aldrich</td><td>SLBR6660V</td><td></td></tr><tr><td>Triton</td><td>Sigma-Aldrich</td><td>9002-93-1</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>White Glass Charged Microscope Slide, 25 x 75 mm Size, Ground Edges, Blue Frosted</td><td>Globe Scientific</td><td>1358D</td><td></td></tr></tbody></table>

identificatie van de bron van uitgescheiden eiwitten in de nier door brefeldin a-injectie

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Identificatie van de bron van uitgescheiden eiwitten in de nier door Brefeldin A-injectie

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes ...

identification-source-secreted-proteins-kidney-brefeldin

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De zebravis volwassen nier is een uitstekend systeem voor renale regeneratie en ziekte studies. Een essentieel aspect van dit onderzoek is de beoordeling van de nefron structuur en functie. Dit protocol beschrijft een aantal methoden die kunnen worden toegepast om nefron tubule samenstelling te beoordelen en om de renale reabsorptie evalueren.

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Weefsel-engineered renale constructies bieden een oplossing voor het tekort aan organen en de schadelijke effecten van dialyse. Hier beschrijven we een protocol bij micro ontleden lymfkliertest nieren voor isolatie van cortico-Wallenberg segmenten. Deze segmenten zijn steiger-vrije cellulaire constructies, vorming van renale organoids ingeplant.

Microdissection van primaire renale weefsel segmenten en integratie met nieuwe steiger-vrije constructie technologie

Dit protocol beschrijft een volledige nier werk-up die moet worden uitgevoerd in muismodellen van glomerulaire ziekte. De methoden toestaan voor gedetailleerde mechanistisch, functionele en structurele analyse van glomerulaire functie, die kan worden toegepast op alle Muismodellen van glomerulaire ziekte.

Beoordeling van de nierfunctie in muismodellen van glomerulaire ziekte

Hier presenteren we een protocol voor het lymfkliertest in vivo labelen van glomerulaire cel oppervlakte eiwitten met biotine. Dit protocol bevat informatie over hoe perfuse muis nieren, het isoleren van de glomeruli, en het uitvoeren van endogene immunoprecipitation van de proteïne van belang.

Isolatie van de Glomeruli en In Vivo Labeling van glomerulaire cel oppervlakte-eiwitten

Hier presenteren we een protocol voor het testen van de glomerulaire permeabiliteit in muizen met behulp van een zeer gevoelige, niet-radioactieve Tracer. Deze methode maakt repetitieve urine analyses mogelijk met kleine urine volumes.

Zeer gevoelige meting van de glomerulaire permeabiliteit bij muizen met Fluorescein isothiocyanaat-poly sucrose 70

De combinatie van antilichaam etikettering, optische clearing en geavanceerde Lichtmicroscopie maakt een driedimensionale analyse van complete structuren of organen mogelijk. Hier beschreven is een eenvoudige methode om immunolabeling van dikke nierschijfjes te combineren, optische clearing met ethyl CINNAMAAT, en confocale beeldvorming die visualisatie en kwantificering van driedimensionale nierstructuren mogelijk maakt.

Optische clearing en beeldvorming van Immunolabeled nierweefsel

Het huidige protocol beschrijft een methode om lupusprogressie bij muizen te volgen. Twee aanvullende procedures worden gepresenteerd om lupus nefritis te karakteriseren op basis van celinfiltratie en eiwitafzetting in de nieren.

Analyses van proteïnurie, renale infiltratie van leukocyten en renale afzetting van eiwitten bij lupusgevoelige MRL / lpr-muizen

Het huidige protocol beschrijft de oprichting van een vliezige nefropathie (MN) diermodel, en hoe de remming van Kemeng Fang de apoptose van PODOCYTEN bij MN-ratten vermindert door de PI3K/AKT-signaleringsroute te activeren.

Mechanisme van Kemeng Fang's remming van podocytapoptose bij ratten met membraneuze nefropathie via de PI3K/AKT-signaleringsroute

Dit protocol beschrijft een methode voor het induceren van unilaterale ureterobstructie (UUO) bij muizen om de progressie van weefselfibrose bij obstructieve nefropathie te bestuderen. Het omvat chirurgische ingrepen, postoperatieve zorg en methoden voor de beoordeling van fibrose.

Unilateraal ureterobstructiemodel voor het onderzoeken van interstitiële fibrose van de nier

Leer een methode met Brefeldin A-injectie om cytokine-producerende cellen in beschadigde nieren te identificeren. Dit protocol maakt kwalitatieve analyse van eiwitten in nierepitheel- en interstitiële cellen mogelijk.

Identificatie van de bron van uitgescheiden eiwitten in de nier door Brefeldin A-injectie

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