High-gennemløb, Multi-Image Cryohistology af mineraliseret væv
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 kræver ofte effektiv fænotypebestemmelse, som ofte er forbundet med en slags histologisk analyse 1-3. Dette behov er endnu mere indlysende med de ambitiøse projekter til at forstyrre hvert gen i mus genom 4. Disse histologiske analyser kan spænde fra vurderingen cellemorfologi og / eller anatomiske træk til kortlægning ekspression af specifikke gener eller proteiner til de enkelte celler. Faktisk er en af de grundlæggende bidrag histologi til genomområdet er evnen til at associere en specifik molekylær signal til en bestemt region eller celletype.
Traditionelle metoder til histologi, især for muskuloskeletale væv, er ofte tidskrævende og besværlig, kræver nogle gange uger til at fastsætte, afkalke, sektion, bejdse, og image prøven derefter analysere billederne via menneskelig fortolkning. Analyse multiple molekylære signaler, enten via immunhistokemi, in situ hybridization, eller specielle pletter, kræver flere sektioner og endda flere prøver til at udføre korrekt. Desuden er disse flere svar kan ikke være co-lokaliseret til den samme celle og undertiden kan ikke være co-lokaliseret til en specifik region i en given prøve. Da genomforskning og Epigenomics felt bevæger sig ind i den digitale tidsalder, skal den histologiske felt også følge trop for at yde effektiv, high-throughput, og automatiseret analyse af en række molekylære signaler inden for en enkelt histologisk snit.
Faktisk er der et behov for forbedrede histologiske teknikker, der kan knytte flere molekylære signaler til specifikke celler inden en given prøve. For nylig har vi udgivet en ny high-throughput cryohistological metode til at vurdere flere respons foranstaltninger inden for en given sektion fra mineraliseret væv 5-14. Processen indebærer stabilisering kryosektionen med frosne cryotape, vedhængende tapede sektion stift til et objektglas, ogudførelse flere runder af farvning og billeddannelse på hver sektion. Disse runder af billeder er derefter justeret manuelt eller via computer automatisering inden billedanalyse (figur 1). Her præsenterer vi detaljerede protokoller i denne proces, og give eksempler, hvor disse teknikker har forbedret vores forståelse af forskellige biologiske processer.
Protocol
Representative Results
Discussion
Her har vi præsenteret en detaljeret cryohistology protokol til at co-lokalisere og kvantificere flere biologiske foranstaltninger ved at tilpasse billeder fra flere runder af farvning / billedbehandling på et enkelt afsnit. Metoden under anvendelse af cryotape er særligt nyttigt, da det opretholder morfologien af vanskelige at vævssnittet (f.eks mineraliseret knogle og brusk). Desuden er det i snit væv klæbet fast til objektglasset, der giver mulighed for flere runder af farvning / billeddannelse af den …
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. |
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