Yüksek Verim, Mineralize Dokuların Çoklu Resim 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
Biyolojik araştırmalar genellikle sık histolojik analiz 1-3 çeşit ile ilişkili verimli fenotiplendirilmesini gerektirir. Bu ihtiyaç, fare genomuna 4 her genin bozmaya daha da belirgin iddialı projelerle olduğunu. Bu histolojik analizler tek tek hücrelere spesifik genlerin veya proteinlerin eşleme ifadesi hücre morfolojisi ve / veya anatomik özellikleri değerlendirmek arasında olabilir. Aslında, genomik alana histoloji temel katkılardan biri, belirli bir bölge ya da hücre tipine özel bir moleküler sinyali ilişkilendirmek yeteneğidir.
Özellikle kas-iskelet dokular için histoloji geleneksel yöntemler, genellikle zaman alıcı ve zahmetli, bazen düzeltmek için hafta gerektiren vardır, decalcify, bölüm, leke ve görüntü numune daha sonra insan yorumuna aracılığıyla görüntüleri analiz. In situ hybridizat edip, immünohistokimya ile, çoklu moleküler sinyallerin incelenmesiiyon ya da özel lekeleri, uygun gerçekleştirmek için birden çok bölüm ve hatta birden fazla örnekleri gerektirir. Ayrıca, bu çoklu tepkiler verilen bir örnekten içinde belirli bir bölgeye eş lokalize edilemez bazen aynı hücreye birlikte lokalize ve olamaz. genomik ve epigenomics alan dijital çağa hareket ettikçe, histolojik alanı da verimli, yüksek verim ve tek histolojik bölüm içinde moleküler sinyallerin çeşitli otomatik analizini sağlamak için davayı takip gerekir.
Gerçekten de, belirli bir numune içindeki belirli hücrelere birden fazla moleküler sinyalleri ilişkilendirmek geliştirilmiş histolojik teknikler için bir talep vardır. Son zamanlarda, mineralize dokusundan 5-14, belirli bir bölüm içinde birkaç yanıt tedbirleri değerlendirmek için yeni bir yüksek verimli cryohistological yöntemi yayınlamıştır. işlem, bir mikroskop lamı katı bantlanmış bölümüne yapışan, dondurulmuş cryotape ile cryosection stabilize içerir veHer bölümünde boyama ve görüntüleme birkaç tur yürütülmesi. Görüntülerin bu tur daha sonra elle veya görüntü analiziyle önce bilgisayar otomasyon (Şekil 1) ile hizalanır. Burada, bu sürecin ayrıntılı protokolleri sunmak ve bu tekniklerin değişik biyolojik süreçlerin anlayışımızı geliştirdik örnekler sunmak.
Protocol
Representative Results
Discussion
Burada birlikte lokalize ve tek bir bölüm üzerinde lekelenme / görüntüleme birden mermi görüntüleri hizalayarak çeşitli biyolojik önlemlerin ölçmek için detaylı bir cryohistology protokolü sundu. Bu bölüm, dokuya güç morfolojisi (örneğin, mineralize edilmiş kemik ve kıkırdak) muhafaza etmesi cryotape kullanılarak belirtilen yöntem özellikle faydalıdır. Buna ek olarak, kesit doku, aynı bölümün boyama / görüntüleme sanmı için izin cam slayt sıkıca yapıştırılır; seri …
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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