Hele Mount Dissektion og Immunofluorescens af Voksen Mouse Cochlea
Summary
Vi præsenterer en metode til at isolere voksne organ Corti som tre intakte cochlear sving (apex, midten og bunden). Vi viser også en procedure for immunfarvning med fluorescens mærkede antistoffer. Tilsammen gør disse teknikker gør det muligt studiet af hårceller, støtteceller, og andre celletyper fundet i cochlea.
Abstract
The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.
Introduction
Den spiralformede cochlea i det indre øre, der er indeholdt i den tidsmæssige knoglen, huser organ Corti, det auditive sensoriske ende orgel i pattedyr. Cochlea er tonotopically organiseret og almindeligt opdelt i apikal, midterste og basal vender svarende til forskellige frekvens regioner med høj frekvens lyd detektion i basen og afsløring lav frekvens i spidsen 1. Hårceller, de mechanosensory celler i organet for Corti, køre længden af cochlea, hvilket er cirka 6 mm lange i mus 2,3. Disse celler konvertere den mekaniske energi af lydbølger, som transmitteres gennem væskefyldte membranøs labyrint, ind i neurale signaler, der behandles af de centrale auditive strukturer. Den her beskrevne teknik tilvejebringer en fremgangsmåde til fremstilling af hele underlag af cortiske organ efter forkalkning af cochlea er færdig (for prøver i området fra en uge gamle til voksenalderen). Vi præsenterer også en fremgangsmåde til immunostalingen hele monteret cochlear væv. Cochlear hele underlag er afgørende for visualisering af alle hårceller og omgivende støtteceller i deres naturlige rumlige arrangementer og muliggøre analyse i tre dimensioner med brug af konfokal mikroskopi.
Drs. Hans Engstrøm og Harlow Ades oprindeligt beskrevet en hel mount cochlear dissektionsmetoden i 1966. De nærmere en teknik til hurtigt at fastsætte og dissekere forkalket cochleae nedsænket i væske fra en række pattedyr, bevare korte intakte segmenter af cortiske organ til mikroskopisk analyse 4. Dissektion af en ikke fastgjort, forkalket rotte cochlea er også blevet vist i en instruktionsvideo 5. Drs. Barbara Bohne og Gary Harding ved Washington University gjort flere vigtige ændringer til denne metode. I deres version af den cochlear hele mount metode, tindingebenet var afkalket, indlejret i plast, og fem halve drejninger eller ti kvart-sving var dissecTed 6,7. Dr. Charles Liberman og kolleger på Eaton Peabody Laboratories, Massachusetts øjet og øret Ambulatorium, ændret denne teknik, så plastik indlejring ikke var forpligtet 8. Yderligere modifikation af teknikken opstod i Dr. Jian Zuo laboratorium på St. Jude Children Research Hospital 9-12, som informerede dissektion metoden præsenteres her. Vi bruger en anden strategi for at få adgang til organ Corti end Bohne og Liberman, som giver mulighed for isolering af komplet apikal, midterste og basale sving. Det dissekerede væv er således større og mindre tilbøjelige til at blive tabt eller beskadiget under dissektion eller Immunofarvning processer. Hertil kommer, at nuværende metode letter måling af afstanden fra den apikale spids eller basal krog til at identificere et frekvensområde.
Selv om mange laboratorier udfører immunfarvning af cochlear væv, er det uklart, hvor denne metode stammer fra. Som følge heraf er der forskellige opskrifter til blokering buffers og antistof inkubation buffere, der kan påvirke udviklingen i de enkelte primære antistoffer. Her præsenterer vi en metode til immunfarvning med fluorescens mærkede antistoffer, der er gældende for de fleste almindeligt anvendte antistoffer i det auditive område.
Den komplekse form af cochlea, sarte struktur af cortiske organ, og benet indkapsling tilvejebringe en udfordring for histologiske og biokemiske analyser. En række teknikker er for tiden anvendes i hørelse felt at overvinde disse vanskelige egenskaber og visualisere cellerne inden organet for Corti, hver teknik med sine egne fordele og ulemper. Protokollen præsenteres her giver mulighed for hele mount dissektion af voksne mus cochlea, og med let modifikation, kan potentielt anvendes til at undersøge de kritiske strukturer i cochleae fra en række andre organismer, der anvendes model på området.
Protocol
Representative Results
Discussion
Der er flere kritiske trin for en vellykket helhed mount dissektion og immunfarvning. Men før en af disse metoder udføres, er nødvendig korrekt fiksering af cochlear væv. Vi anbefaler at bruge methanol gratis, ultrarent, EM kvalitet PFA. PFA fremstillet af pulver kan have spor af methanol og en ustabil pH, som nedsætter kvaliteten af immunofluorescens. Andre grupper har også vist, at lignende dissektioner er mulige ved hjælp af fikseringsmidler, som ikke indeholder formaldehyd 14-16. …
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by a grant from the Office of Naval Research (N000141310569). The Southern Illinois University School of Medicine Research Imaging Facility equipment was supported by the National Center for Research Resources-Health (S10RR027716).
Materials
| Standard pattern forceps | Fine Science Tools | 11000-12 | can be purchased from other vendors |
| 10.5 cm fine scissors | Fine Science Tools | 14060-11 | can be purchased from other vendors |
| 2 ml microcentrifuge tubes | MidSci | AVSS2000 | can be purchased from other vendors |
| 16% formaldehyde, methanol free, ultra pure, EM grade | Polysciences | 18814 | TOXIC –wear gloves and cannot be disposed of in the sink. Can be purchased from other vendors. |
| PBS pH 7.4 | Sigma | P3813-10PAK | can be purchased from other vendors |
| EDTA | Fisher | BP118-500 | can be purchased from other vendors |
| end-over-end tube rotator | Fisher | 05-450-127 | can be purchased from other vendors |
| 60 mm petri dish | Fisher | 50-202-037 | can be purchased from other vendors |
| Dow Corning Sylgard 184 silicone encapsulant kit | Ellsworth Adhesives | 184 SIL ELAST KIT 0.5KG | |
| activated charcoal | Fisher | AC134372500 | can be purchased from other vendors |
| stereo dissection microscope | Zeiss | Stemi 2000 | can be purchased from other vendors |
| Dumont #4 jeweler's forceps | Fine Science Tools | 11241-30 | |
| Dumont #5 jeweler's forceps | Fine Science Tools | 11251-20 | |
| 2.5 mm Vannas spring scissors | Fine Science Tools | 15001-08 | curved |
| 5 mm Vannas-Tubingen spring scissors | Fine Science Tools | 15003-08 | straight |
| 48 well plates | Fisher | 08-772-52 | can be purchased from other vendors |
| 8 well chamber slides | Fisher | 1256518 | can be purchased from other vendors |
| Triton X-100 | Sigma | X100-500 | can be purchased from other vendors |
| BSA, fraction V | Fisher | BP1605 | can be purchased from other vendors |
| NGS | Vector labs | S-1000 | can be purchased from other vendors |
| NHS | Vector labs | S-2000 | can be purchased from other vendors |
| 3D rotator | Midsci | R3D-710 | can be purchased from other vendors |
| Western blot incubation box XL | Licor | 929-97401 | |
| Hoechst 33342 | Life Technologies | H3570 | can be purchased from other vendors |
| Prolong gold antifade mounting media | Life Technologies | P36930 | can be purchased from other vendors,but mounting medias vary in their ability to protect against photobleaching |
| Superfrost Plus Slides | Fisher | 12-550-15 | can be purchased from other vendors |
| coverslips 22 x 22 x 1 | Fisher | 12-548-B | can be purchased from other vendors |
| clear nail polish | Local drug store | can be purchased from other vendors | |
| cardboard slide folder | Fisher | 12-587-10 | can be purchased from other vendors |
| plastic slide box | Fisher | 03-448-10 | can be purchased from other vendors |
Referenzen
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