Name: tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones
Uploaded: 2012-01-26T12:00:00
Description: Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones at JoVE.com

Vi takker Dr. Yuriy M. Usachev for hans hjælp i den indledende standardisering af konfokal mikroskopi / billeder af mus plantar punch biopsi immunfarvning, og Dr. Donna L. Hammond for hendes fortsatte hjælp og konstruktiv kritik i dette arbejde. Dette arbejde blev finansieret af tilskud fra NINDS / NIH (NS069898), og en Idéudvikling Grant Award fra Department of Defense Prostate Cancer Research Program (DoD-PCRP-101096) til DPM

1. Animal perfusion Alle dyreforsøg udføres i denne undersøgelse er godkendt af Institutional Animal Care og brug udvalg fra University of Iowa, og følg NIH retningslinier for brug af dyr i forskningsøjemed. <ol><li> På dagen før perfusion, forberede 1 L af fosfat buffer (0,2 M PB i dobbelt destilleret H 2 O, pH 7,4), og opbevares ved 4 ° C. Dette vil blive anvendt til perfusion og post-fiksering processer. </li><li> På dagen for perfusion, forberede 500 ml 4,0% parafor...

<ol>
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Her har vi detaljeret de metoder til udarbejdelse af mus hud-og knoglevæv sektioner for immunfarvning og afsløring af perifere sensoriske nervefibre. Afsnittene fremstillet af plantar hudbiopsier indeholder både glatte og behårede hud, hvilket betyder, at protokollen kan bruges på enhver hudtype. Disse teknikker kan også anvendes til farvning af andre celletyper i disse væv (fx leukocytter, vaskulær endothelia, glatte muskelceller blandt andre). Disse metoder giver et glimrende kompromis mellem optimale ultrastr...

<table border="1"><tbody><tr><td> Materialets navn </td><td> Type </td><td> Firma </td><td> Katalog Nr. </td><td> Kommentar </td></tr><tr><td> 3mm Harris Micro-Punch </td><td> Materiale </td><td> Ted Pella </td><td> 15094 </td><td></td></tr><tr><td> Perfusion </td><td> Materiale </td><td> VWR International </td><td> 23609-170 </td><td></td></tr><tr><td> Paraformaldehyd </td><td> Reagens </td><td> Fisher Scientific </td><td> T353 </td><td></td></tr><tr><td> Pikrinsyre </td><td> Reagens </td><td> Sigma-Aldrich </td><td> 239801 </td><td></td></tr><tr><td> Oktober Indlejring sammensatte </td><td> Reagens </td><td> Tissue-Tek </td><td> 4583 </td><td></td></tr><tr><td> CYTO-Freeze kryogene aerosol spray </td><td>Materiale </td><td> Kontrol Firma </td><td> 3118 </td><td></td></tr><tr><td> Goat Serum </td><td> Reagens </td><td> Sigma-Aldrich </td><td> G9023 </td><td></td></tr><tr><td> Inkubation bakke og låg til immunfarvning (Large) </td><td> Materiale </td><td> RPI Corp </td><td> 248270 (bakke) 248270-A (låg) </td><td></td></tr><tr><td> ImmEdge vandafvisende barriere pen </td><td> Materiale </td><td> Vector Laboratories </td><td> H-4000 </td><td></td></tr><tr><td> Kamelhår Børster (# 1 tykkelse) </td><td> Materiale </td><td> Ted Pella </td><td> 11859 </td><td></td></tr><tr><td> Pro-Long Gold Mounting Medium </td><td> Reagens </td><td> Invitrogen </td><td> P36930 </td><td></td></tr></tbody></table>

tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones

Detection og primær behandling af fysiske, kemiske og termiske sensoriske stimuli af perifere sensoriske nervefibre er nøglen til sansning i dyr og mennesker. Disse perifere sensoriske nervefibre udtrykke et væld af receptorer og ion-kanal proteiner, som registrerer og iværksætte specifikke sensoriske stimuli. Der findes metoder til karakterisere de elektriske egenskaber af perifere sensoriske nervefibre innerverer huden, som også kan bruges til at identificere funktionelle udtryk for specifikke ion-kanal proteiner i disse fibre. Men lignende elektrofysiologiske metoder er ikke tilgængelige (og er også vanskeligt at udvikle) til påvisning af den funktionelle udtryk af receptorer og ion-kanal proteiner i perifere sensoriske nervefibre innerverer andre indre organer, herunder de mest udfordrende væv såsom knogle. Desuden kan sådanne elektrofysiologiske metoder ikke anvendes til at bestemme et udtryk for ikke-overgearet proteinS i perifere sensoriske nervefibre. Derfor immunfarvning af perifere / viscerale vævsprøver for sensoriske nerve Håndbold giver den bedst mulige måde til at bestemme et udtryk for specifikke proteiner af interesse i disse nervefibre. Hidtil har de fleste protein udtryk studier i sensoriske neuroner udnyttet immunfarvning procedurer i sensoriske ganglier, hvor oplysningerne er begrænset til ekspression af specifikke proteiner i cellen kroppen af ​​bestemte typer eller delmængder af sensoriske neuroner. Her rapporterer vi detaljerede metoder / protokoller for udarbejdelsen af ​​perifere / viscerale vævsprøver for immunfarvning af perifere sensoriske nervefibre. Vi har specielt detalje metoder til forberedelse af huden eller plantar punch biopsi og knogler (femur) afsnit fra mus til immunfarvning af perifere sensoriske nervefibre. Disse metoder er ikke kun nøglen til kvalitativ bestemmelse af protein udtryk i perifere sensoriske neuroner, men også give en kvantitativ analyse metode tilafgørende ændringer i protein udtryk niveauer i bestemte typer eller delmængder af sensoriske fibre, samt til bestemmelse af morfologiske og / eller anatomiske ændringer i antallet og tætheden af ​​sensoriske fibre under forskellige patologiske tilstande. Desuden er disse metoder ikke begrænset til farvning af kun sensoriske nervefibre, men kan også bruges til farvning alle typer af nerve fibre i huden, knogler og andre viscerale væv.

Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones at JoVE.com

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Immuncytokemiske identifikation af perifer sensorisk nerve fiber undertyper (og påvisning af protein udtryk deri) er nøglen til forståelsen af ​​molekylære mekanismer, der ligger perifert sensation. Her har vi beskrive metoder til udarbejdelse af perifere / viscerale vævsprøver, såsom hud og lemmer knogler, for specifikke immunfarvning af perifere sensoriske nervefibre.

Tissue Forberedelse og immunfarvning af Mouse sensoriske nervefibre innerverer hud og lemmer Bones

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