Name: visualizing the actin and microtubule cytoskeletons at the b-cell immune synapse using stimulated emission depletion (sted) microscopy
Uploaded: 2018-04-09T14:00:00
Description: Watch this Scientific Journal Video about Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy at JoVE.com

Vi takker UBC Life Sciences Institute (LSI) Imaging facilitet for at støtte og vedligeholde STED mikroskop. Dette arbejde blev finansieret af tilskud #68865 fra den canadiske institutter for sundhedsforskning (til M.R.G.). Vi takker Dr. Kozo Kaibuchi (Nagoya University, Nagoya, Japan) for klip-170-NGL plasmidet.

Alle dyr procedurer blev godkendt af University of British Columbia dyr efterbehandling udvalget.
1. at give udtryk for normal god landbrugspraksis-fusion proteiner i A20 B-lymfom celler
<ol><li>Kultur A20 celler i komplet RPMI medium (RPMI-1640 suppleret med 10% varme-inaktiverede føtal bovint serum (FBS), 50 µM 2-mercaptoethanol, 2 mM glutamin, 1 mM pyruvat, 50 U/mL penicillin og 50 µg/mL streptomycin) ved 37 ° C i en vævskultur kuvøse med 5 % CO2.</li>
	<li>Forberede Tran...

<ol>
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Detaljerede billeder af cytoskeletal strukturer kan opnås ved hjælp af STED super-resolution mikroskopi, der teoretisk set kan opnå en opløsning på 50 nm, i forhold til konventionelle Konfokal mikroskopi, hvor diffraktion-begrænset opløsning er ~ 200 nm 24. evnen til at løse finere strukturer er yderligere forbedret ved hjælp af deconvolution software til at beregne den mest sandsynlige placering af den oprindelige lyskilde fra det observerede "slørede" fluorescens signal. Denne protokol...

Når B-celler bindes til polariseret arrays af antigener (f.eks.vises på overfladen af antigen-præsentere celler (PMV'er)), den resulterende B-celle receptoren (BCR) signalering drev dannelsen af en klassisk immun synapse struktur, som først blev beskrevet i T celler1,2,3,4,5,6,7,8,9,10, 11,12,13. I første omgang, microclusters af antigen-bundet oplysningspligt form i periferien af B celle: APC kontakt site. Disse microclusters derefter bevæge sig mod midten af antigen kontakt site, hvor de hånd i hånd i en central Supramolekylær aktivering klynge (cSMAC), der udgør kernen i den immun synapse. Immun synapse dannelse optimerer BCR signalering og letter BCR-medieret antigen udvinding fra APC membran14. Denne antigen erhvervelse, som efterfølges af BCR-medieret antigen internalisering og efterfølgende antigen behandling, giver mulighed for B-celler til at præsentere peptid: MHC II komplekser til T-celler og fremkalde T-celle hjælp14. Fordi immun synapse dannelse fremmer B celle aktivering, belyse de mekanismer, der etablerer denne funktionelle mønster af receptor organisation kan give nye er indsigt i hvordan LUN immun svar indledt og reguleret.
Reorganisering af både aktin og mikrotubulus cytoskeletons er afgørende for immun synapse dannelse. Lokaliseret BCR signalering stimuleret af et rumligt polariseret vifte af antigener inducerer hurtige og dramatiske remodellering af actin cytoskeleton1,15. Dannelsen af dendritiske actin strukturer i periferien af cellen B udøver skubbe styrker på plasma membran og fremmer spredning af B-celle. Dette giver mulighed for B-celle til at scanne et større område af antigen-bærende overflade, og øger antallet af referencematerialer, der binder antigen og aktivere BCR signalering veje. På samme tid er MTOC og mikrotubulus netværk omlagt til stedet af antigen kontakt. Da MTOC tilgange antigen kontakt site, udvide mikrotubuli stammer fra MTOC langs indersiden af plasma membran på grænsefladen mellem B-celle og antigen-bærende overflade16,17. Disse juxtamembrane mikrotubuler kan derefter fungere som spor til de dynein-medieret centripetale bevægelse af antigen-bundet BCR microclusters18, fører til dannelsen af en cSMAC.
Omlægning og polarisering af MTOC mod immun synapse kræver intakt aktin og mikrotubulus cytoskeletons, og ofte afhænger af samspillet mellem den kortikale actin netværk og mikrotubuli16,17, 19,20. Kortikale actin-bindende proteiner, såsom IQGAP1, kan fange mikrotubuli ved at interagere med proteinkomplekser, at dekorere mikrotubulus plus-slutter21. Disse dynamiske komplekser af plus-end bindende proteiner omfatter EB1 og klip-170, som benævnes kollektivt som mikrotubulus plus-ende tracking proteiner (+ TIPs)21,22. + TIPs i enderne af mikrotubuli kan binder sig til proteiner, der er forbundet med enten plasma membran eller kortikale actin cytoskelet. Dette giver kraft-genererende mekanismer (f.eks.minus-ende instrueret bevægelse af cortically forankret dynein langs mikrotubuli) at udøve trække styrker på mikrotubuli, og dermed flytte MTOC. CLIP-170 kan binde til aktin-associerede stilladser protein IQGAP123, og vi har vist, at begge af disse proteiner er nødvendige for BCR-induceret MTOC polarisering mod immun synapse17. Denne IQGAP1-klip-170 interaktion kan spille en central rolle i koordineringen af ombygning af actin cytoskeleton med Repositionering af mikrotubulus nettet på B-celle immun synapse.
Konventionelle Fluorescens mikroskopi har afsløret den dramatiske reorganisering af actin og mikrotubulus cytoskeletons under B-celle immun synapse dannelse2. Men denne fremgangsmåde ikke kan løse små cellulære strukturer i detaljer på grund af diffraktion grænse af lys, som ifølge Abbes lov, er afhængig af bølgelængde af lys bruges til at belyse prøven og blænde af de objektive24. Denne diffraktion grænse begrænser opløsning af konventionelle lys mikroskoper til 200-300 nm i lateral retning og 500-700 nm i aksial retning25. Derfor, mindre subcellulært strukturer, som de fine detaljer af actin og mikrotubulus cytoskeletons, kunne kun konstateres ved hjælp af elektronmikroskopi. Elektronmikroskopi billeddannelse af cytoskelettet er tidskrævende, kræver hård prøve fiksering og forberedelse protokoller, der kan ændre biologiske strukturer, og er begrænset til antistof-medieret påvisning. Mulighed for at immunostain og samtidigt billede flere proteiner eller cellulære strukturer er en væsentlig fordel ved Fluorescens mikroskopi. Desuden udtrykker fluorescerende fusion proteiner i cellerne giver mulighed for real-time imaging og er nyttig, når effektive antistoffer for immunfarvning protein af interesse ikke er tilgængelige.
Seneste teknologiske fremskridt i Super-resolution mikroskopi har overvundet diffraktion grænserne af lys og tilladt visualisering af nanoskala cellestrukturer24. En sådan super-resolution mikroskopi teknik kaldes stimuleret emission udtynding (STED) mikroskopi. STED beskæftiger to lasere, hvor en laser ophidser fluorophore og en anden laser med en doughnut-formet mønster selektivt undertrykker fluorescens emission omkring fluorophore. Dette reducerer funktionen punkt-spredning (tilsyneladende område) af en enkelt partikel, fluorescerende og giver en sub diffraktion grænse fluorescerende billede25,26. Grundtilstand udtynding mikroskopi beskæftiger også fluorescens-baserede teknikker til at erhverve super-resolution billeder. Dog billede erhvervelse og genopbygning gange er lange, der er kun et begrænset antal fluorophores, der kan bruges, og den samtidige høj opløsning billeddannelse af flere cytoskeletal komponenter teknisk udfordrende fordi opretholde aktin og mikrotubulus strukturer kræver forskellige fiksering procedurer. Således STED har flere fordele over elektronmikroskopi og andre super-resolution mikroskopi nærmer sig, det tilbyder hurtig billede erhvervelse, har minimal post-processing krav, og beskæftiger det samme fluorophores og farvning teknikker der anvendes til konventionelle Fluorescens mikroskopi af faste prøver26.
Super-resolution mikroskopi er nu blevet brugt til at visualisere actin strukturer på den immun synapse i natural killer (NK) celler og T celler26,27,28,29,30, 31. dog super-resolution billeddannelse af mikrotubulus cytoskeleton, samt koordineret reorganisering af actin og mikrotubulus cytoskeletons under immune synapse dannelse, har først for nylig blevet rapporteret17. Vi brugte STED mikroskopi billede B celler, der havde fået lov til at sprede på coverslips belagt med anti-immunglobulin (anti-Ig) antistoffer, som stimulerer BCR signalering og indlede cytoskeleton reorganisering. Når belagt på immobiliserede anti-Ig antistoffer, gennemgå B-celler dramatiske actin-afhængige spredning, som sammenfatter de indledende events under immune synapse dannelse. Vigtigere, afslørede STED mikroskopi de fine detaljer af dendritiske ring af F-actin, der former i periferien af immun synapse og viste, at MTOC samt mikrotubuli knyttet til det, havde flyttet tæt på antigen kontakt site17. Disse mikrotubuli udvides udad mod den perifere ring af F-actin. Derudover flerfarvet STED billeddannelse af forskellige kombinationer af F-actin, tubulin, IQGAP1, og normal god landbrugspraksis-tagged klip-170 + TIPs viste at mikrotubulus plus-ends præget af CLIP-170-normal god landbrugspraksis var tæt forbundet med den perifere actin meshwork og med IQGAP1, en kortikale capture protein17.
Vi præsenterer her, en detaljeret protokol for imaging aktin og mikrotubulus cytoskeletons på immun synapse ved hjælp af STED mikroskopi. Disse metoder har været optimeret ved hjælp af linjen A20 murine B-celle, som er blevet bredt ansat til at studere BCR signalering og immun synapse dannelse17,32,33,34,35 , 36 , 37 , 38 , 39. fordi kommercielle antistoffer mod klip-170 ikke virkede godt for immunfarvning i tidligere forsøg, vi beskriver i detaljer udtrykket for normal god landbrugspraksis-tagged klip-170 i A20 celler, sammen med farvning protokoller for samtidig visualisere op til tre cytoskeletal komponenter eller cytoskeleton-associerede proteiner. Metoder til brug af STED mikroskopi til billede actin på NK celle immun synapser har været beskrevet tidligere40. Her, udvider vi det til hentning af multi-farve Super-opløsning billeder af både aktin og mikrotubulus cytoskeletons i B-celler.
En kritisk overvejelse for Super-resolution mikroskopi ved hjælp af passende fiksering procedurer for vedligeholdelse af cellestrukturer og at forebygge skade fluorescerende proteiner. Fiksering og farvning metoder præsenteres heri er blevet optimeret for at opretholde normal god landbrugspraksis fluorescens og giver høj opløsning billeddannelse af actin- og mikrotubulus. Når at udtrykke fluorescerende proteiner, skal det bemærkes, at B-celler er normalt vanskelige at transfect. Ved hjælp af denne protokol, 20-50% af A20 celler typisk express transfected normal god landbrugspraksis fusion protein, og denne befolkningen niveauer af protein udtryk er variabel. Ikke desto mindre super-resolution billeddannelse af actin og mikrotubuli ved hjælp af de procedurer, der beskriver vi er ganske robust og høj kvalitet billeder opnås let. På trods af deres lille størrelse i forhold til A20 celler viser vi, at disse procedurer kan også bruges til at afbilde mikrotubulus netværk i primære B-celler, der har været kortvarigt aktiveret med LPS (LPS). Vi har vist, at LPS-aktiveret primær B-celler kan være transfekteret med siRNAs ved relativt høj effektivitet (dvs., sådan at protein nedbrydning kan påvises ved immunoblotting), hvilket gør dem et godt alternativ til brugen af B-cellelinier til nogle undersøgelser 17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A20 mouse B-lymphoma cells</td>
			<td>ATCC</td>
			<td>TIB-208</td>
			<td>Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td>Heat inactivate at 56 oC for 30 min</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Millipore Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Millipore Sigma</td>
			<td>G5763</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Millipore Sigma</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Liquid, 10,000 units</td>
		</tr>
		<tr>
			<td>Additional materials for primary B cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#181;m cell strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic bead-based B cell isolation kit</td>
			<td>Stemcell Technologies</td>
			<td>19854</td>
			<td>EasySep Mouse B cell Isolation kit</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS)</td>
			<td>Millipore Sigma</td>
			<td>L4391</td>
			<td>LPS from E. coli 0111:B4</td>
		</tr>
		<tr>
			<td>B cell-activating factor (BAFF)</td>
			<td>R&#38;D Systems</td>
			<td>2106-BF-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid encoding CLIP-170-GFP</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. Kozo Kaibuchi (Nagoya Univ., Nagoya, Japan); described in Fukata et al. Cell 109:873-885, 2002</td>
		</tr>
		<tr>
			<td>Recommended transfection reagents and equipment </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa Nucleofection kit V</td>
			<td>Lonza</td>
			<td>VCA-1003</td>
			<td>Follow the manufacturer&#39;s directions for mixing the transfection reagents with the DNA</td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector model 2b</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td>Program L-013 used</td>
		</tr>
		<tr>
			<td>Falcon 6-well plates, TC treated, sterile</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Coating coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18-mm diameter round #1.5 cover glasses</td>
			<td>Thomas Scientific</td>
			<td>1217N81</td>
			<td>Similar product: Marienfield-Superior catalogue #117580</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>1381242</td>
			<td>Dissecting extra fine splinter forceps</td>
		</tr>
		<tr>
			<td>Falcon 12-well sterile tissue polystyrene tissue culture plate</td>
			<td>Corning</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>100% methanol</td>
			<td>Fisher Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile phosphate buffered saline (PBS) without calcium or magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti-mouse IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-008</td>
			<td>For A20 cells</td>
		</tr>
		<tr>
			<td>Goat-anti-mouse IgM antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-020</td>
			<td>For primary mouse B cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% stock solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Dilute with PBS to working concentration</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (50% stock solution)</td>
			<td>Millipore Sigma</td>
			<td>340855</td>
			<td>Dilute with PBS to working concentration</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Millipore Sigma</td>
			<td>S2149</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), Fraction V</td>
			<td>Millipore Sigma</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab52866</td>
			<td>1:250 dilution recommended but should be optimized</td>
		</tr>
		<tr>
			<td>Alexa Fluor 532-conjugated goat anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11009</td>
			<td>1:100 dilution recommended but should be optimized</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568-conjugated phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12380</td>
			<td>1:100 dilution recommneded but should be optimized</td>
		</tr>
		<tr>
			<td>Prolong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36970</td>
			<td>Without DAPI</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>P1150-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Millipore Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Millipore Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 TCS STED microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Huygens deconvolution software</td>
			<td>Scientific Voume Imaging</td>
			<td></td>
			<td>See https://svi.nl/HuygensProducts</td>
		</tr>
	</tbody>
</table>

visualizing the actin and microtubule cytoskeletons at the b-cell immune synapse using stimulated emission depletion (sted) microscopy

B-celler, der bindes til membran-bundet antigener (fx, på overfladen af en antigen-præsenterer celle) udgør en immun synapse, en specialiseret cellestruktur, der optimerer B-celle receptoren (BCR) signalering og BCR-medieret antigen erhvervelse. Begge remodellering af actin cytoskelettet og omlægning af mikrotubulus netværk mod antigenet kontakt site er afgørende for immun synapse dannelse. Ombygning af actin cytoskeleton i en tætte perifere ring af F-actin er ledsaget af polarisering af den organisering af mikrotubulus center mod immun synapse. Mikrotubulus plus-end bindende proteiner samt kortikale plus-ende capture proteiner mægle fysiske vekselvirkninger mellem actin og mikrotubulus cytoskeletons, som tillader dem at blive reorganiseret på en koordineret måde. Belyse de mekanismer at kontrollere denne cytoskeletal reorganisering, samt forstå hvordan disse cytoskeletal strukturer forme immun synapse dannelse og BCR signalering, kan give ny indsigt i B-celle aktivering. Dette har været hjulpet af udviklingen af super-resolution mikroskopi metoder, der afslører nye detaljer om cytoskeletal netværksorganisation. Vi beskriver her en metode for at anvende stimuleret emission udtynding (STED) mikroskopi til samtidigt billede actin strukturer, mikrotubuli og transfekteret normal god landbrugspraksis-tagged mikrotubulus plus-end bindende proteiner i B-celler. Til model de tidlige begivenheder i immun synapse dannelse, tillader vi B-celler til at sprede på coverslips belagt med anti-immunglobulin (anti-Ig) antistoffer, der indleder BCR signalering og cytoskeleton remodellering. Vi leverer en trinvis protokoller for at give udtryk for normal god landbrugspraksis fusion proteiner i A20 B-lymfom celler, for anti-Ig-induceret celle spredning og for efterfølgende celle fiksering, immunfarvning, image erhvervelse og billede deconvolution trin. Billeder med høj opløsning fremstillet ved hjælp af disse procedurer, at man kan samtidig visualisere actin strukturer, mikrotubuli og mikrotubulus plus-end bindende proteiner, der kan forbinde disse to cytoskeletal netværk.

For B-celler spredes på immobiliserede anti-Ig, giver STED mikroskopi anvendes i forbindelse med deconvolution software billeder i højere opløsning af cytoskeletal strukturer end Konfokal mikroskopi. Dette er tydeligt i figur 1, hvor F-actin netværk blev visualiseret ved hjælp af den protokol, der er beskrevet ovenfor. En sammenligning af Konfokal og STED super-resolution billeder af den samme prøve viser, at STED billederne er af højere opløsning og ...

Watch this Scientific Journal Video about Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy at JoVE.com

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

Vi præsenterer en protokol for at bruge STED mikroskopi til samtidigt billede actin strukturer, mikrotubuli og mikrotubulus plus-end bindende proteiner i B-celler, der har spredt på coverslips belagt med antistoffer til B-celle receptoren, en model for den indledende fase af immun synapse dannelse.

Visualisere aktin og mikrotubulus Cytoskeletons på B-celle immun Synapse ved hjælp af stimuleret Emission udtynding (STED) mikroskopi

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

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Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

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Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

Visualisere makrofag ekstracellulære fælder ved hjælp af konfokalmikroskopi

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Co farvning blodkar og Nerve fibre i fedtvæv

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...