高通量，矿化组织的多图像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
生物学研究通常需要高效率的表型，这是经常用某种组织学分析1-3相关联。这需要的是更为明显的雄心勃勃的计划，扰乱小鼠基因组中4每个基因。这些组织学分析的范围可以从评估细胞形态和/或解剖特征来特定基因或蛋白质到单个细胞的映射表达式。实际上，组织学的基因组学领域中的基本贡献之一是为特定的分子信号到特定区域或细胞类型相关联的能力。
组织学的传统方法，尤其是对肌肉骨骼组织中，往往费时费力，有时需要数周来修复时间，脱钙，部分染色和图像标本然后分析通过人眼可识别图像。分析多个分子信号，通过免疫组化，是否在原地 hybridizat离子或特殊污渍，需要多个章节，甚至多个标本适当地执行。此外，这些多个响应无法被共定位到相同的细胞，有时不能共定位到特定的区域的给定样品中。随着基因组学和表观基因组学领域的移动进入数字化时代，组织学领域也必须跟进，提供高效，高通量，以及单个组织切片中的各种分子信号的自动分析。
事实上，存在对可以在给定样品中的特定小区中的多个分子信号关联改进的组织学技术的需求。最近，我们已经发布了从矿化组织5-14评估某一部分内的几个应对措施的新高通量方法cryohistological。该过程涉及稳定冷冻cryotape的冷冻切片，拘泥于所录部分显微镜幻灯片，开展几轮染色和成像上的每节。这些轮次的图像，然后手动或通过之前的图像分析计算机自动化（ 图1）对齐。在这里，我们提出这一过程的详细协议的，并提供这些技术提高了我们不同的生物过程的理解的例子。
Protocol
Representative Results
Discussion
这里，我们已经提出了详细cryohistology协议共定位并通过在一个单一的部对准来自多轮染色/成像的图像量化几个生物措施。因为它维持的困难部分组织的形态（ 例如 ，矿化骨和软骨）使用cryotape概述的方法是特别有用的。此外，该截面组织被牢固地附着于玻璃载片，允许多轮同一节染色/成像的;不像在那里连续切片分别染色，不同的协议传统的方法。采用连续切片会带来一个问题，尝?…
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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