Name: quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections
Uploaded: 2017-07-31T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

Forfatterne takker dr. Fahad Bafakih og Jim Mandell til erhvervelse og identifikation af patientprøver, Garrett F. Beeghly til bistand med immunhistokemi og Biorepository and Tissue Research Facility, Histology Core for Cardiovascular Research Center og Biomolecular Analysis Facility ved University of Virginia for at få hjælp til Prøveopkøb, immunhistokemi og billeddannelse.

Denne protokol identificerer cellulære komponenter i formalin-fikserede paraffinindlejrede (FFPE) prøver, som det er typisk for bankede kliniske patientprøver. Paraffin indlejring giver mulighed for den bedste vedligeholdelse af cellulær og vævs morfologi samt har bedre levetid af sektioner. De prøver, der blev brugt til denne analyse, blev adgang til via University of Virginia Biorepository og Tissue Research Facility. Patientprøver blev udvalgt af en neuropatolog baseret på en endelig diagnose af glioblastom (...

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Vores metode foreslået her er en kvantitativ tilgang til analyse af histologiske prøver farvet ved anvendelse af traditionel kromogen immunhistokemi. Nuværende metode til denne type analyse indeholder lignende farvningsprotokoller efterfulgt af gradering af uafhængige patologer. Denne metode har været pålidelig, men for en række anvendelser kræves en mere præcis forståelse af cellulær sminke, såsom bedre forståelse af heterogeniteten forbundet med tumorer og nøjagtig rekapitulation af tumorer til in vitro-...

Glioblastom (GBM), den mest almindelige og ondartede hjernecancer, er præget af en meget diffus invasion fra den primære tumorbulb i den omgivende sunde hjerneparenchym 1 , 2 . Denne diffuse invasion gør tumoren særligt vanskelig at resektere fuldt ud, og de invaderende kræftceller, der forbliver post-terapi, er den mest almindelige årsag til uundgåelig gentagelse 2 , 3 , 4 . Tidligere fandt vi, at den diffuse gliomcelleinvasion er inhiberet for at være terapeutisk gavnlig 5 , men der er ikke meget kendt om de komplekse mekanismer, som bidrager til GBM-invasionen. Tumormiljømiljøet eller vævet omkring kræften har været impliceret i progressionen af ​​tumorer i flere kræftformer 6 , 7 . Glioblastom tumor mikromiljøet, på sArtikulær, er relativt underkarakteriseret og er entydigt kompleks, sammensætning af flere gliaceller, såsom astrocytter, microglia og oligodendrocytter, såvel som ekstracellulær matrix, opløselige faktorer og biofysiske faktorer. Eksperimentelt har astrocytter og microglia vist sig at øge gliomprogressionen og invasionen 8 , 9 , 10 , men sammensætningen af ​​alle glialceller i den native humane hjerne mikromiljø er ukendt. Vi tidligere viste mikromiljøkomponenter kan forudse patientoverlevelse ved kvantitativt at analysere cellulære komponenter i glioblastom mikromiljøet og inkorporere vores analyser i en proportional fare model 11 . Her beskriver vi den kvantitative analysemetode til identifikation af populationerne af glialceller i tumorvolumenet og tilstødende regioner af tumorresektioner fra glioblastompasientens. Vi anvendte kromogen immunhistokemi for at identificere glialcellepopulationerne og ImageJ for at analysere procentdækning af farvning for hver glialpopulation. Vurdering af procentdækning skaber en simpel måling til bestemmelse af de morfologiske forskelle i celler, især dem, der påvirkes af interaktioner med kræftceller. Tidligere undersøgelser til kvantificering af histopatologisk farvning anvender standardfarvning, såsom hæmatoxylin og eosin 12 eller Massons trichrom 13 , som ikke udnytter specificiteten af ​​antistofbaseret immunhistokemist farvning. Vores metode blev udviklet til direkte kvantificering af glialpopulationerne inden for glioblastom-patienttumorresektioner, som vi tilstræber at bruge til at belyse det komplekse glioblastom-mikromiljø.

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			<td height="42" style="height:42px;width:216px;">Xylene</td>
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			<td height="42" style="height:42px;width:216px;">High pH antigen unmasking solution</td>
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			<td>H-3301</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">TBS</td>
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			<td height="21" style="height:21px;width:216px;">Triton-X</td>
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			<td style="width:119px;">9002-93-1</td>
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			<td height="21" style="height:21px;width:216px;">Horse serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">Anti-ALDH1L1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab56777</td>
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			<td height="21" style="height:21px;width:216px;">Anti-Iba1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab5076</td>
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			<td height="42" style="height:42px;width:216px;">Anti-Oligodendrocyte Specific Protein1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab53041</td>
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			<td height="42" style="height:42px;width:216px;">ImmPRESS anti-goat</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">MP-7405</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">ImmPRESS Universal (anti-mouse/rabbit)</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">MP-7500</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Hydrogen peroxide</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">216763</td>
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			<td height="42" style="height:42px;width:216px;">ImmPACT DAB substrate</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">SK-4105</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Hematoxylin counterstain</td>
			<td style="width:71px;">ThermoScientific</td>
			<td style="width:119px;">72404</td>
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			<td height="21" style="height:21px;width:216px;">Histochoice Mounting Media</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">H157-475</td>
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			<td height="63" style="height:63px;width:216px;">Aperio Scanscope</td>
			<td style="width:71px;">Leica Biosystems</td>
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			<td height="63" style="height:63px;width:216px;">Image Scanscope</td>
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			<td height="84" style="height:84px;width:216px;">Super HT PAP Pen</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">195506</td>
			<td></td>
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quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections

Med den voksende interesse for tumor mikromiljøet satte vi op for at udvikle en metode til specifikt at bestemme mikromiljøkomponenterne inden for patientprøver af glioblastom, den dødeligste og mest invasive hjernekræft. Ikke alene er kvantitative metoder til gavn for nøjagtigt at beskrive syge væv, de kan også potentielt bidrage til mere nøjagtig prognose, diagnose og udvikling af vævsmonterede systemer og udskiftninger. I glioblastom er glialceller, såsom microglia og astrocytter, uafhængigt korreleret med dårlig prognose baseret på patologisk klassificering. Tilstanden af ​​disse celler og andre glialcellekomponenter er imidlertid ikke blevet beskrevet godt kvantitativt. Dette kan være svært på grund af de store processer, der markerer disse glialceller. Desuden fokuserer de fleste histologiske analyser på den samlede vævsprøve eller kun inden for størstedelen af ​​tumoren, i modsætning til at afgrænse kvantifikationer baseret på regioner hvideHin det meget heterogene væv. Her beskriver vi en metode til at identificere og kvantitativt analysere populationerne af glialceller inden for tumorvolumen og tilstødende regioner af tumorresektioner fra glioblastompatienter. Vi anvendte kromogen immunhistokemi til at identificere glialcellepopulationerne i patienttumorresektioner og ImageJ for at analysere procentdækning af farvning for hver glialpopulation. Med disse teknikker er vi i stand til bedre at beskrive glialcellerne i regionerne af gliomastomikromiljøet.

Til denne analyse blev to interessegrupper inden for vores tumorresektioner - den primære tumorvolumen og de tilstødende regioner, primært sammensat af sundt væv med diffus invaderende kræftceller ( figur 1A , 1B ) - identificeret af samarbejdende neuropatologer på hæmatoxylin og eosinfarvet patient prøver. Inden for hver patientprøve identificeredes positiv farvning for astrocytter ( figur 1C ), microg...

Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Kvantitativ immunhistokemi af det cellulære mikromiljø i patientglioblastomresektioner

Denne protokol blev udviklet til kvantitativt at identificere tumor mikro-miljøkomponenter i glioblastom patientresektioner ved anvendelse af kromogen immunhistokemi og ImageJ.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

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