높은 처리량, 광물 조직의 멀티 이미지 Cryohistology
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
생물학적 연구는 종종 자주 조직 학적 분석 1-3 일종의와 관련된 효율적인 표현형가 필요합니다. 이 필요 마우스 게놈 4 각 유전자를 방해 더욱 분명 야심 찬 프로젝트가있다. 이러한 조직 학적 분석은 개별 세포에 특정 유전자 또는 단백질의 매핑 표현 세포 형태 및 / 또는 해부학 적 특징을 평가에 이르기까지 다양 할 수 있습니다. 사실, 유전체 분야에 조직학의 기본적인 기여 중 하나가 특정 영역 또는 세포 유형으로 특정 분자 신호를 연결하는 기능이다.
특히 근골격계 조직에 대한 조직 검사의 전통적인 방법은 종종 시간이 많이 걸리고 힘들고, 때로는 해결하기 위해 주를 필요로하고, 석회, 섹션, 얼룩 및 이미지 시편은 인간의 해석을 통해 이미지를 분석 할 수 있습니다. 시츄 hybridizat의 여부 면역 통해 여러 분자 신호를 분석이온, 또는 특수 얼룩, 적절하게 수행하기 위해 여러 섹션, 심지어 여러 표본을 필요로한다. 또한, 이러한 복수 응답은 주어진 시료 내 특정 지역에 공동 지역화 할 수 없습니다 때로는 동일한 셀에 공동 지역화 할 수 없습니다. 게놈과 후성 유전체 매핑 센터, 후성 유전체학 분야는 디지털 시대로 이동함에 따라, 조직 학적 필드는 효율적이고 높은 처리량, 단일 조직 학적 섹션 내 분자 신호의 다양한 자동화 된 분석을 제공하기 위해 소송을 수행해야합니다.
실제로, 소정의 시험편의 특정 셀에 다수의 분자 신호들을 연결할 수 향상 조직학 기술에 대한 필요성이 대두되고있다. 최근에, 우리는 광물 조직 5-14에서 주어진 섹션 내에서 여러 가지 대응 방안을 평가하기위한 새로운 높은 처리량 cryohistological 방법을 발표했다. 방법은 현미경 슬라이드에 견고하게 접착 테이프 부 냉동 cryotape로 동결 절단 (Cryosections) 안정화 포함하며각 섹션에 염색 및 이미징의 여러 라운드를 실시. 이미지의 이러한 라운드는 수동 또는 이미지 분석하기 전에 컴퓨터 자동화 (그림 1)을 통해 정렬됩니다. 여기, 우리는이 과정에 대한 자세한 프로토콜을 제시하고 이러한 기술은 서로 다른 생물학적 과정에 대한 우리의 이해를 향상 예를 제공합니다.
Protocol
Representative Results
Discussion
여기에서 우리는 공동 현지화 및 단일 섹션에 염색 / 영상의 여러 라운드에서 이미지를 정렬하여 여러 가지 생물학적 조치를 정량화하기위한 세부 cryohistology 프로토콜을 제시 하였다. 이 섹션 조직에 어려운 형태 (예를 들어, 광물 뼈와 연골을) 유지로 cryotape을 사용하여 설명하는 방법은 특히 유용합니다. 또한, 단면 조직은 동일한 부의 염색 / 영상 여러 발사를 허용 유리 슬라이드에 단단?…
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. |
Referências
- Schofield, P. N., Vogel, P., Gkoutos, G. V., Sundberg, J. P. Exploring the elephant: histopathology in high-throughput phenotyping of mutant mice. Dis Model Mech. 5 (1), 19-25 (2012).
- Adissu, H. A., et al. Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen. Disease Models and Mechanisms. 7 (5), 515-524 (2014).
- Johnson, J. T., et al. Virtual histology of transgenic mouse embryos for high-throughput phenotyping. PLoS Genet. 2 (4), e61 (2006).
- Ayadi, A., et al. Mouse large-scale phenotyping initiatives: overview of the European Mouse Disease Clinic (EUMODIC) and of the Wellcome Trust Sanger Institute Mouse Genetics Project. Mamm.Genome. 23 (9-10), 600-610 (2012).
- Utreja, A., et al. Cell and matrix response of temporomandibular cartilage to mechanical loading. Osteoarthr Cartil. 24 (2), 335-344 (2016).
- Dyment, N. A., et al. Gdf5 progenitors give rise to fibrocartilage cells that mineralize via hedgehog signaling to form the zonal enthesis. Dev.Biol. 405 (1), 96-107 (2015).
- Lalley, A. L., et al. Improved biomechanical and biological outcomes in the MRL/MpJ murine strain following a full-length patellar tendon injury. J.Orthop.Res. 33 (11), 1693-1703 (2015).
- Breidenbach, A. P., et al. Ablating hedgehog signaling in tenocytes during development impairs biomechanics and matrix organization of the adult murine patellar tendon enthesis. J.Orthop.Res. 33 (8), 1142-1151 (2015).
- Ushiku, C., Adams, D., Jiang, X., Wang, L., Rowe, D. Long Bone Fracture Repair in Mice Harboring GFP Reporters for Cells within the Osteoblastic Lineage. J.Orthop.Res. 28 (10), 1338-1347 (2010).
- Matthews, B. G., et al. Analysis of asMA-labeled progenitor cell commitment identifies notch signaling as an important pathway in fracture healing. J.Bone Miner.Res. 29 (5), 1283-1294 (2014).
- Dyment, N. A., Hagiwara, Y., Jiang, X., Huang, J., Adams, D. J., Rowe, D. W. Response of knee fibrocartilage to joint destabilization. Osteoarthritis Cartilage. 23 (6), 996-1006 (2015).
- Grcevic, D., et al. In vivo fate mapping identifies mesenchymal progenitor cells. Stem Cells. 30 (2), 187-196 (2012).
- Hong, S. H., Jiang, X., Chen, L., Josh, P., Shin, D. G., Rowe, D. Computer-Automated Static, Dynamic and Cellular Bone Histomorphometry. J Tissue Sci Eng. Suppl 1, 004 (2012).
- Matthews, B. G., Torreggiani, E., Roeder, E., Matic, I., Grcevic, D., Kalajzic, I. Osteogenic potential of alpha smooth muscle actin expressing muscle resident progenitor cells. Bone. 84, 69-77 (2016).
- Hagiwara, Y., et al. Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair. J.Orthop.Res. 33 (7), 948-956 (2015).
- Jiang, X., et al. Histological analysis of GFP expression in murine bone. J.Histochem.Cytochem. 53 (5), 593-602 (2005).
- Nissanov, J., Bertrand, L., Tretiak, O. Cryosectioning distortion reduction using tape support. Microsc.Res.Tech. 53 (3), 239-240 (2001).
- Kawamoto, T. Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch.Histol.Cytol. 66 (2), 123-143 (2003).
