À haut débit, multi-images Cryohistology de minéralisés Tissues
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
La recherche biologique nécessite souvent phénotypage efficace, qui est souvent associée à une sorte d'analyse histologique 1-3. Ce besoin est encore plus évident avec les projets ambitieux de perturber chaque gène dans le génome de la souris 4. Ces analyses histologiques peuvent varier d'évaluer la morphologie des cellules et / ou des caractéristiques anatomiques de l'expression de la cartographie de gènes ou de protéines spécifiques aux cellules individuelles. En fait, l'un des apports fondamentaux de l'histologie au domaine de la génomique est la possibilité d'associer un signal moléculaire spécifique à une région ou d'une cellule de type spécifique.
Les méthodes traditionnelles de l'histologie, en particulier pour les tissus musculo-squelettiques, sont souvent longue et laborieuse, nécessitant parfois des semaines pour fixer, détartrer, section, tache, et l'image de l'échantillon, puis d'analyser les images via l'interprétation humaine. L' analyse des signaux moléculaires multiples, que ce soit par immunohistochimie, hybridizat in situion, ou des colorations spéciales, nécessite de multiples sections et même des échantillons multiples pour effectuer de manière appropriée. En outre, ces réponses multiples ne peuvent pas être co-localisés à la même cellule et parfois ne peuvent pas être co-localisée à une région spécifique dans un échantillon donné. Comme le domaine de la génomique et épigénomique se déplace dans l'ère numérique, le champ histologique doit également suivre pour fournir efficace, à haut débit, et l'analyse automatisée d'une variété de signaux moléculaires dans une coupe histologique unique.
En effet, il existe une demande pour une amélioration des techniques histologiques qui peuvent associer des signaux moléculaires multiples à des cellules spécifiques dans un échantillon donné. Récemment, nous avons publié un nouveau haut-débit méthode cryohistological pour évaluer plusieurs mesures d'intervention au sein d' une section donnée de tissu minéralisé 5-14. Le procédé consiste à stabiliser la cryosection avec cryotape congelé, faire adhérer l'article collé de façon rigide sur une lame de microscope, etla réalisation de plusieurs cycles de coloration et de l'imagerie sur chaque section. Ces séries d'images sont ensuite alignées manuellement ou grâce à l' automatisation de l' ordinateur avant l'analyse de l' image (figure 1). Ici, nous présentons des protocoles détaillés de ce processus et de fournir des exemples où ces techniques ont amélioré notre compréhension des différents processus biologiques.
Protocol
Representative Results
Discussion
Ici, nous avons présenté un protocole de cryohistology détaillé pour co-localiser et quantifier plusieurs mesures biologiques en alignant les images provenant de plusieurs cycles de coloration / imagerie sur une seule section. La méthode décrite à l' aide de la cryotape est particulièrement utile car il conserve la morphologie des tissus difficiles à la section (par exemple, os minéralisé et le cartilage). En outre, le tissu sectionné adhère fermement à la lame de verre, ce qui permet plusieur…
Declarações
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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