Name: plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy
Uploaded: 2017-05-05T12:30:00
Description: Watch this Scientific Journal Video about Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy at JoVE.com

Vi udtrykker vores taknemmelighed til M. Bouchecareilh, E. Tétaud, S. Duvezin-Caubet, og A. Devin for deres hjælp og kommentarer til manuskriptet. Vi er taknemmelige for den elektroniske billedbehandling pol af Bordeaux Imaging Center, hvor billederne blev taget. Dette arbejde blev støttet af Centre National de Recherche Scientifique.

1. Fremstilling af Formvar Grid Film BEMÆRK: Udfør chloroform manipulation under en emhætte bruger personlige værnemidler (handsker, kittel, briller). Anvende 400 mesh elektronmikroskopi kobbergitre. Med andre grid typer (andre maskestørrelser, guld og nikkel gitre), kvaliteten af ​​frysning er værre. <ol><li> Fremstilling af en 100 ml opløsning af 0,3% formvar i chloroform. Lade den blive opløst natten over uden omrøring. </li><li> Sætte 400 mesh (antal huller per kvadrattom...

<ol>
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TEM er en kraftfuld metode til ultrastrukturelle observation af organeller, celler og væv. Cryofixation / fryse-substitution er i øjeblikket den bedste metode til konservering af både ultrastruktur og protein antigenicitet. Kemiske fikseringsmidler trænge ind og virke meget langsomt hvorved strukturelle omlejringer før fuldstændig stabilisering af ultrastrukturen 2. Omvendt cryofixation / fryse-substitution øjeblikkeligt stabiliserer cellulære strukturer 4. Dog cry...

Prøve forberedelse er afgørende for succes i enhver elektronmikroskopi undersøgelse. Konventionel EM fiksering var den primære metode til fastsættelse væv eller celler til transmissionselektronmikroskopi (TEM) 1. Først aldehyder og osmiumtetroxid anvendes til kemisk fastgøre materialet ved stuetemperatur. Derefter er materialet dehydreret med organiske opløsningsmidler, infiltreret og indlejret i epoxyharpiks. Denne metode er afhængig af dækningsgraden fikseringer i cellen. Følgelig er artefakter og ekstraktion af cellulære indhold observeres sædvanligvis 2. Cryofixation er klart en bedre alternativ til konservering af cellulære strukturer 3, holde dem intakt. Kan opnås den højeste kvalitet af TEM billeder 4 i tynde harpiksafsnit hjælp af cryofixation / fryse substitution metode. Formålet med denne teknik er at opnå forglasset biological prøver uden iskrystaller dannelse eller indeholder iskrystaller små nok til ikke at beskadige ultrastruktur af cellerne. Højtryks-frysning (HPF) og ultrahurtig cryofixation, som også kaldes springet frysning (PF), er to metoder til cryofix prøver. HPF immobiliserer molekyler i cellen går og undgår skader forårsaget af konventionel EM fiksering. Flere typer af frysemaskiner og automatiske substitutionsreaktioner indretninger er blevet udviklet 5. Indefrysningsforanstaltningerne maskiner og hjælpematerialer (flydende nitrogen, specimen bærere, etc.) er dyre, men de giver mulighed for fremstilling af høj kvalitet elektronmikrografier 6, 7. PF er en teknik, som blev anvendt i begyndelsen af 1950&#39;erne og er beskrevet i litteraturen som enkel og billig 8 .Under PF fremgangsmåde udføres den forbehandlede prøve frosset ved en hurtig hastighed til opnåelse af iskrystaller på mindre end 3 og 5 nm. Til dette formål er prøvenkastet ud i en flydende kryogen, såsom ethan, propan, eller en ethan-propan-blanding. Siden 1950&#39;erne har forbedringer af PF blevet gjort for at gøre denne teknik til rådighed for et større antal brugere. Højtryks-frysning er i øjeblikket den eneste måde, hvorpå at fryse en lang række prøver tykkere end 50 um (op til en tykkelse på 200 um for skiveformede prøver) 5, mens PF vidt omfang anvendes til at afbilde små genstande (&lt;100 nm ), såsom makromolekylære komplekser suspenderet i en tynd film af amorf is 9. Større prøver, fx eukaryote celler, kan cryofixed af PF, men kræver prøveholder, såsom kapillær kobberrør eller sandwichsystemer 8, 10, 11. Her, en hurtig, nem at bruge og billig springet frysning / fryse-substitution teknologi kan anvendes til forskellige suspension cellekulturer præsenteres.

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plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy

Transmission Electron Microscopy (TEM) er et ekstraordinært redskab til at studere celle ultrastruktur, for at lokalisere proteiner og visualisere makromolekylære komplekser med meget høj opløsning. Men for at komme så tæt som muligt på den naturlige tilstand, er perfekt prøve konservering påkrævet. Konventionel elektronmikroskopi (EM) fiksering med aldehyder, for eksempel, giver ikke god ultrastrukturel konservering. Den langsomme penetration fikseringer inducerer celle reorganisering og tab af forskellige cellekomponenter. Derfor er konventionelle EM fiksering ikke mulighed for en øjeblikkelig stabilisering og konservering af strukturer og antigenicitet. Det bedste valg for behandlingen intracellulære begivenheder er at bruge cryofixation efterfulgt af fryse-substitution fiksering metode, der holder cellerne i deres native tilstand. Højtryks-frysning / fryse-substitution, som bevarer integriteten af ​​cellulær ultrastruktur, er den mest almindeligt anvendte metode, men kræver expensive udstyr. Her er en nem at bruge og billig fryse fiksering metode efterfulgt af fryse-substitution for suspension cellekulturer fremlagt.

I denne artikel er en nem at bruge og billig springet frysning fremgangsmåde til ultrastrukturelle (figur 1 og figur 2) og immunmærkning (figur 3) undersøgelser fremlagt. Vi viser, at det ikke er nødvendigt at have særligt udstyr til fryse-substitution proceduren, og at opvarmningen procedure tager mindre end 6 timer i stedet for de 24 timer ved hjælp af et dedikeret system. <p class="jove...

Watch this Scientific Journal Video about Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy at JoVE.com

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Dette manuskript beskriver en nem at bruge og billig cryofixation fremgangsmåde til visualisering suspensionsceller ved transmissionselektronmikroskopi.

Plunge Nedfrysning: Et værktøj for Ultrastrukturelle og immunlokalisering Studier af Suspension Celler i Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

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Biology

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