Summary

Høy gjennomstrømming, Multi-Image Cryohistology av mineralisert vev

Published: September 14, 2016
doi:

Summary

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

Abstract

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

Introduction

Biologisk forskning krever ofte effektiv fenotyping, som er ofte forbundet med noen form for histologisk analyse 1-3. Dette behovet er enda mer tydelig med de ambisiøse prosjekter for å forstyrre hvert gen i musegenomet 4. Disse histologiske analyser kan være alt fra å vurdere celle morfologi og / eller anatomiske trekk til kartlegging uttrykket av spesifikke gener eller proteiner til individuelle celler. Faktisk er en av de grunnleggende bidragene fra histologi til feltet av genom evne til å knytte en spesifikk molekyl signal til en spesifikk region eller celletype.

Tradisjonelle metoder for histologi, spesielt for muskel vev, er ofte tidkrevende og arbeidskrevende, krever noen ganger uker for å fikse, avkalke, seksjon, beis, og bilde prøven deretter analysere bildene via menneskelig fortolkning. Analysere flere molekylære signaler, enten via immunhistokjemi, in situ hybridization, eller spesielle flekker, krever flere seksjoner og enda flere prøver å utføre riktig. I tillegg er disse flere svar kan ikke være co-lokalisert til samme celle, og noen ganger kan ikke være co-lokalisert til et bestemt område i et gitt mønster. Som genom og Epigenomics felt beveger seg inn i den digitale tidsalder, må den histologiske feltet også følge etter for å gi høy ytelse og høy gjennomstrømming, og automatisk analyse av en rekke forskjellige molekylære signaler i en enkelt histologisk seksjon.

Faktisk er det et behov for forbedrede histologiske teknikker som kan knytte flere molekylære signaler til spesifikke celler i en gitt prøve. Nylig har vi publisert en ny high-throughput cryohistological metode for å vurdere flere responstiltak innenfor en gitt seksjon fra mineralisert vev 5-14. Prosessen innebærer å stabilisere cryosection med frosne cryotape, man følger den teipet delen fast til et objektglass, oggjennomføre flere runder med flekker og bildebehandling på hver del. Disse rundene av bildene blir deretter justert manuelt eller via datamaskin automatisering før bildeanalyse (figur 1). Her presenterer vi detaljerte protokoller av denne prosessen, og gi eksempler hvor disse teknikkene har forbedret vår forståelse av ulike biologiske prosesser.

Protocol

The University of Connecticut Health Center institusjonelle dyr omsorg og bruk komité godkjente alle dyre prosedyrer. 1. Fiksering og Inkludering Avlive dyret via CO 2 kvelning eller andre godkjente metoder. Høste vev av interesse (f.eks lem, ryggvirvler, etc.) og plasser i 10% nøytral bufret formalin ved 4 ° C inntil skikkelig festet. Vær spesielt nøye med å opprettholde konsistent anatomisk plassering før fiksering. For eksempel, fikse lemme…

Representative Results

En generell Arbeidsflyt for høy gjennomstrømming, Multi-Image Cryohistology Figur 1 viser den generelle arbeidsflyten som brukes for denne teknikken. Det inkluderer flere trinn fra fiksering gjennom flere runder med bildebehandling og endelig bildejusteringen / analyse. Prosessen kan ta så lite som en uke å gå fra prøve fiksering gjennom 4 runder med bildebehandling, som er kortere tid e…

Discussion

Her har vi presentert en detaljert cryohistology protokoll til co-lokalisere og kvantifisere flere biologiske tiltak ved å samkjøre bilder fra flere runder med farging / bildebehandling på en enkelt seksjon. Fremgangsmåten som er beskrevet ved hjelp av cryotape er spesielt nyttig som det opprettholder morfologi vanskelig å seksjon vev (f.eks mineralisert ben og brusk). I tillegg er seksjonert vev klebet fast til glass-slide, slik at for multiple runder med farging / avbildning av den samme delen; i motsetn…

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to acknowledge the following funding sources: NIH R01-AR063702, R21-AR064941, K99-AR067283, and T90-DE021989.

Materials

10% neutral buffered formalin Sigma Aldrich HT501128-4L Multiple suppliers available. Toxic. Can be substituted with 4% paraformaldehyde.
Sucrose Sigma Aldrich S9378 Multiple suppliers available.
PBS Sigma Aldrich P5368 Multiple suppliers available.
Cryomolds Fisher Scientific Fisherbrand #22-363-554 Different sizes can be used depending on tissue
Cryomatrix Thermo Scientific 6769006 Can be substitituted with other cryomatrices. However, PVA/PEG-based resins have worked best in our hands.
2-methyl-butane Sigma Aldrich M32631 Multiple suppliers available.
Cryostat Leica Biosystems 3050s Can be substituted with other brands/models.
Specimen disc Leica Biosystems 14037008587 Can be substituted with other brands/models.
Cryostat blades Thermo Scientific 3051835 Can be substituted with other brands/models.
Cryotape Section Lab Cryofilm 2C
Roller Electron Microscopy Sciences 62800-46 Can be substituted with other brands/models.
Plastic microscope slides Electron Microscopy Sciences 71890-01 Can be substituted with other brands/models.
Glass microscope slides Thermo Scientific 3051 Can be substituted with other brands/models.
Norland Optical Adhesive, 61 Norland Optical Norland Optical Adhesive, 61
UV Black Light General Electric F15T8-BLB
Glacial acetic acid Sigma Aldrich ARK2183 Multiple suppliers available.
Chitosan Sigma Aldrich C3646 Multiple suppliers available.
InSpeck red microscopheres ThermoFisher Scientific I-14787
InSpeck green microspheres ThermoFisher Scientific I-14785
Calcein Blue Sigma Aldrich M1255 Multiple suppliers available.
Calcein Sigma Aldrich C0875  Multiple suppliers available.
Alizarin complexone Sigma Aldrich A3882  Multiple suppliers available.
Demeclocycline Sigma Aldrich D6140  Multiple suppliers available.
NaHCO3 Sigma Aldrich S5761 Multiple suppliers available.
Glycerol Sigma Aldrich G5516 Multiple suppliers available.
ELF 97 yellow fluorescent acid phosphatase substrate ThermoFisher Scientific E-6588
DAPI ThermoFisher Scientific 62247 Multiple suppliers available. Can be substituted with Hoechst 33342 or other nuclear dyes.
TO-PRO-3 (Cy5 nuclear counterstain) ThermoFisher Scientific T3605
Propidium Iodide ThermoFisher Scientific R37108 Multiple suppliers available.
Sodium acetate anhydrous Sigma Aldrich S2889 Multiple suppliers available.
sodium tartrate dibasic dihydrate  Sigma Aldrich T6521 Multiple suppliers available.
Sodium nitrite Sigma Aldrich S2252 Multiple suppliers available.
Tris Sigma Aldrich 15504 Multiple suppliers available.
MgCl2 hexahydrate Sigma Aldrich M9272 Multiple suppliers available.
NaCl Sigma Aldrich S7653 Multiple suppliers available.
Fast Red TR Salt Sigma Aldrich F8764 Multiple suppliers available. Can also be substituted with other substrate kits such as Vector Blue (Vector Laboratories, Cat# SK-5300)
Naphthol AS-MX  Sigma Aldrich N4875 Multiple suppliers available.
N,N dimethylformamide Sigma Aldrich D158550 Multiple suppliers available.
Toluidine blue O Sigma Aldrich T3260 Multiple suppliers available.
Axio Scan.Z1 Carl Zeiss AG Axio Scan.Z1 Other linear or tiled scanners may also be used.
DAPI Filter Set Chroma Technology Corp. 49000
CFP Filter Set Chroma Technology Corp. 49001
GFP Filter Set Chroma Technology Corp. 49020
YFP Filter Set Chroma Technology Corp. 49003
Custom yellow (ELF 97) Filter Set Chroma Technology Corp. custom; HQ409sp, HQ555/30m, 425dxcr
TRITC Filter Set Chroma Technology Corp. 49004
Cy5 Filter Set Chroma Technology Corp. 49009
CryoJane Tape Transfer System Electron Microscopy Sciences 62800-10 Multiple suppliers available.
CryoJane Tape Windows Electron Microscopy Sciences 62800-72 Multiple suppliers available.
CryoJane Adhesive Slides Electron Microscopy Sciences 62800-4X Multiple suppliers available.

References

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Cite This Article
Dyment, N. A., Jiang, X., Chen, L., Hong, S., Adams, D. J., Ackert-Bicknell, C., Shin, D., Rowe, D. W. High-Throughput, Multi-Image Cryohistology of Mineralized Tissues. J. Vis. Exp. (115), e54468, doi:10.3791/54468 (2016).

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