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 uttrykker vår takknemlighet til M. Bouchecareilh, E. Tétaud, S. Duvezin-Caubet og A. Devin for deres hjelp og kommentarer til manuskriptet. Vi er takknemlige for den elektroniske billedkolonnen i Bordeaux Imaging Center hvor bildene ble tatt. Dette arbeidet ble støttet av Center National de la Recherche Scientifique.

1. Fremstilling av Formvar Grid Film MERK: Utfør kloroform manipulasjon under en avtrekkshette ved hjelp av personlig verneutstyr (hansker, frakk, briller). Bruk 400 mesh elektronmikroskopi kobbernett. Med andre nett-typer (andre maskestørrelser, gull og nikkel rutenett), kvaliteten av frysing er verre. <ol><li> Fremstill en 100 ml oppløsning av 0,3% Formvar i kloroform. La den løse over natten uten omrøring. </li><li> Sett 400 mesh (antall hull per kvadrattomme) gitrene elektron micr...

<ol>
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TEM er en kraftig metode for ultra observasjon av organeller, celler og vev. Cryofixation / fryse-substitusjonen er i dag den beste metode for bevaring av både ultrastruktur og protein antigenisitet. Kjemiske fiksativer penetrere og virke meget sakte og derved tillate strukturelle rearrangementer før den fullstendige stabilisering av ultra 2. Omvendt, cryofixation / fryse-substitusjon vendte stabiliserer cellestrukturer 4. Imidlertid cryofixation / fryse-substitusjons te...

Prøvepreparering er avgjørende for suksess for et hvilket som helst elektronmikroskopi studien. Konvensjonelle EM fiksering var den primære metode for fiksering av vev eller celler for transmisjons-elektronmikroskopi (TEM) 1. Først blir aldehyder og osmium-tetroksyd som brukes for kjemisk å feste materialet ved romtemperatur. Deretter blir materialet dehydrert med organiske oppløsningsmidler, infiltrert og innstøpt i epoksyharpiks. Denne metode er avhengig av inntrengningshastigheten for bindemiddel inn i cellen. Følgelig blir gjenstander og utvinning av celleinnholdet som vanligvis observeres 2. Cryofixation er helt klart et bedre alternativ for konservering av cellulære strukturer 3, holde dem intakt. Den høyeste kvalitet av TEM-bilder 4 i tynne harpiks seksjoner kan oppnås ved bruk av cryofixation / fryse-substitusjonsmetoder. Hensikten med denne teknikk er å oppnå forglasset biological samplene uten iskrystaller dannes eller som inneholder iskrystaller som er små nok til ikke å skade den ultrastrukturen av cellene. Høytrykks frysing (HPF) og ultra-hurtig cryofixation, som også kalles stupe frysing (PF), er to fremgangsmåter for å cryofix prøver. HPF immobiliserer molekyler i cellen umiddelbart og unngår skader forårsaket av konvensjonell EM fiksering. Flere typer av frysing maskiner og automatiske substitusjonsinnretninger er blitt utviklet 5. Fryse maskiner og materialer (flytende nitrogen, prøven bærere, etc.) er kostbare, men de gir mulighet for produksjon av høykvalitets-elektronmikros 6, 7. PF er en teknikk som ble brukt i de tidlige 1950-tallet og er omtalt i litteraturen så enkel og billig 8 .During PF prosedyre, blir den preparerte prøven frosset ved en hurtig hastighet for å oppnå iskrystaller størrelse mindre enn 3 til 5 nm. For å oppnå dette, er utvalgetkastet ut i et flytende kryogen, så som etan, propan, eller et etan-propan-blanding. Siden 1950-tallet har forbedringer av PF blitt gjort for å gjøre denne teknikken tilgjengelig for et større antall brukere. Høytrykks frysing er for tiden den eneste mulige måten å fryse en stor rekke prøver tykkere enn 50 um (opp til en tykkelse på 200 pm for skiveformede prøver) 5, mens PF er mye brukt til bilde små gjenstander (&lt;100 nm ), slik som makromolekylære komplekser suspendert i en tynn film av amorft is 9. Større prøver, for eksempel eukaryote celler, kan cryofixed av PF, men krever prøveholdere, slik som kapillar kobberrør eller sandwich-systemer 8, 10, 11. Her, en rask, lett å bruke og rimelig stupe frysing / fryse-substitusjon teknologi som kan anvendes for forskjellige suspensjon cellekulturer er presentert.

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			<td height="21" style="height:21px;width:215px;">Grids</td>
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plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy

Transmisjonselektronmikroskopi (TEM) er en ekstraordinær verktøy for å studere celle ultrastrukturen, for å lokalisere proteiner og visualisere makromolekylære komplekser med meget høy oppløsning. Men for å komme så nær som mulig til den opprinnelige tilstand, perfekt prøve bevaring nødvendig. Konvensjonell elektronmikroskopi (EM) fiksering med aldehyder, for eksempel, gir ikke god ultra konservering. Den langsomme gjennomtrengning av bindemiddel induserer celle reorganisering og tap av forskjellige cellekomponenter. Derfor vil konvensjonell EM fiksering ikke tillater en umiddelbar stabilisering og konservering av strukturer og antigenisitet. Det beste valget for behandlingen av intracellulære hendelser er å bruke cryofixation etterfulgt av frysesubstitusjonsfikseringsmetode som holder cellene i deres native tilstand. Høytrykks frysing / fryse-substitusjon, noe som bevarer integriteten av cellulær ultrastrukturen, er den mest brukte metoden, men krever expensive utstyr. Her blir en lett-å-bruke og rimelig frysefikseringsmetode fulgt av fryse-substitusjon for suspensjon cellekulturer presentert.

I denne artikkelen er en lett-å-bruke og rimelig stupe frysemetode for ultra (figurene 1 og figur 2) og immunmerking (figur 3) studier presentert. Vi viser at det ikke er nødvendig å ha spesialutstyr for fryse-substitusjon prosedyre og at oppvarmingen prosedyren tar mindre enn 6 timer i stedet for 24 timer ved hjelp av et eget system. ...

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 manuskriptet beskriver en lett-å-bruke og rimelig cryofixation metode for visualisering av suspensjonsceller av transmisjonselektronmikroskopi.

Plunge Frysing: Et verktøy for Ultra og Immunolocalization Studier av suspensjonsceller i transmisjonselektronmikroskopi

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

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Biology

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

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Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Eksempel forberedelse og bildebehandling for Exosomes av overføring elektronmikroskop

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

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Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

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The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

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Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Rask frysing ved hjelp av sandwichfrysingsenhet for god ultrastrukturell bevaring av biologiske prøver i elektronmikroskopi

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

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