The authors would like to acknowledge the following funding sources: NIH R01-AR063702, R21-AR064941, K99-AR067283, and T90-DE021989.

The University of Connecticut Health Center institutional animal care and use committee approved all animal procedures.
1. Fixation and Embedding
<ol>
	<li>Euthanize the animal via CO2 asphyxiation or other approved methods.</li>
	<li>Harvest the tissue of interest (e.g., limb, vertebrae, etc.) and place in 10% neutral buffered formalin at 4 &#176;C until properly fixed. Take special care to maintain consistent anatomical placement prior to fixation. For instance, fix lim...

<ol>
	<li>Schofield, P. N., Vogel, P., Gkoutos, G. V., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+elephant:+histopathology+in+high-throughput+phenotyping+of+mutant+mice.">Exploring the elephant: histopathology in high-throughput phenotyping of mutant mice.</a> Dis Model Mech. 5 (1), 19-25 (2012).</li><li>Adissu, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+reveals+correlative+and+unique+phenotypes+in+a+high-throughput+mouse+phenotyping+screen.">Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen.</a> Disease Models and Mechanisms. 7 (5), 515-524 (2014).</li><li>Johnson, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+histology+of+transgenic+mouse+embryos+for+high-throughput+phenotyping.">Virtual histology of transgenic mouse embryos for high-throughput phenotyping.</a> PLoS Genet. 2 (4), e61 (2006).</li><li>Ayadi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+large-scale+phenotyping+initiatives:+overview+of+the+European+Mouse+Disease+Clinic+(EUMODIC)+and+of+the+Wellcome+Trust+Sanger+Institute+Mouse+Genetics+Project.">Mouse large-scale phenotyping initiatives: overview of the European Mouse Disease Clinic (EUMODIC) and of the Wellcome Trust Sanger Institute Mouse Genetics Project.</a> Mamm.Genome. 23 (9-10), 600-610 (2012).</li><li>Utreja, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+matrix+response+of+temporomandibular+cartilage+to+mechanical+loading.">Cell and matrix response of temporomandibular cartilage to mechanical loading.</a> Osteoarthr Cartil. 24 (2), 335-344 (2016).</li><li>Dyment, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gdf5+progenitors+give+rise+to+fibrocartilage+cells+that+mineralize+via+hedgehog+signaling+to+form+the+zonal+enthesis.">Gdf5 progenitors give rise to fibrocartilage cells that mineralize via hedgehog signaling to form the zonal enthesis.</a> Dev.Biol. 405 (1), 96-107 (2015).</li><li>Lalley, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+biomechanical+and+biological+outcomes+in+the+MRL/MpJ+murine+strain+following+a+full-length+patellar+tendon+injury.">Improved biomechanical and biological outcomes in the MRL/MpJ murine strain following a full-length patellar tendon injury.</a> J.Orthop.Res. 33 (11), 1693-1703 (2015).</li><li>Breidenbach, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablating+hedgehog+signaling+in+tenocytes+during+development+impairs+biomechanics+and+matrix+organization+of+the+adult+murine+patellar+tendon+enthesis.">Ablating hedgehog signaling in tenocytes during development impairs biomechanics and matrix organization of the adult murine patellar tendon enthesis.</a> J.Orthop.Res. 33 (8), 1142-1151 (2015).</li><li>Ushiku, C., Adams, D., Jiang, X., Wang, L., Rowe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Bone+Fracture+Repair+in+Mice+Harboring+GFP+Reporters+for+Cells+within+the+Osteoblastic+Lineage.">Long Bone Fracture Repair in Mice Harboring GFP Reporters for Cells within the Osteoblastic Lineage.</a> J.Orthop.Res. 28 (10), 1338-1347 (2010).</li><li>Matthews, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+asMA-labeled+progenitor+cell+commitment+identifies+notch+signaling+as+an+important+pathway+in+fracture+healing.">Analysis of asMA-labeled progenitor cell commitment identifies notch signaling as an important pathway in fracture healing.</a> J.Bone Miner.Res. 29 (5), 1283-1294 (2014).</li><li>Dyment, N. A., Hagiwara, Y., Jiang, X., Huang, J., Adams, D. J., Rowe, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+knee+fibrocartilage+to+joint+destabilization.">Response of knee fibrocartilage to joint destabilization.</a> Osteoarthritis Cartilage. 23 (6), 996-1006 (2015).</li><li>Grcevic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fate+mapping+identifies+mesenchymal+progenitor+cells.">In vivo fate mapping identifies mesenchymal progenitor cells.</a> Stem Cells. 30 (2), 187-196 (2012).</li><li>Hong, S. H., Jiang, X., Chen, L., Josh, P., Shin, D. G., Rowe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-Automated+Static,+Dynamic+and+Cellular+Bone+Histomorphometry.">Computer-Automated Static, Dynamic and Cellular Bone Histomorphometry.</a> J Tissue Sci Eng. Suppl 1, 004 (2012).</li><li>Matthews, B. G., Torreggiani, E., Roeder, E., Matic, I., Grcevic, D., Kalajzic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+potential+of+alpha+smooth+muscle+actin+expressing+muscle+resident+progenitor+cells.">Osteogenic potential of alpha smooth muscle actin expressing muscle resident progenitor cells.</a> Bone. 84, 69-77 (2016).</li><li>Hagiwara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+stability+dictates+the+differentiation+pathway+of+periosteal+progenitor+cells+in+fracture+repair.">Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.</a> J.Orthop.Res. 33 (7), 948-956 (2015).</li><li>Jiang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+GFP+expression+in+murine+bone.">Histological analysis of GFP expression in murine bone.</a> J.Histochem.Cytochem. 53 (5), 593-602 (2005).</li><li>Nissanov, J., Bertrand, L., Tretiak, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+distortion+reduction+using+tape+support.">Cryosectioning distortion reduction using tape support.</a> Microsc.Res.Tech. 53 (3), 239-240 (2001).</li><li>Kawamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+new+adhesive+film+for+the+preparation+of+multi-purpose+fresh-frozen+sections+from+hard+tissues,+whole-animals,+insects+and+plants.">Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants.</a> Arch.Histol.Cytol. 66 (2), 123-143 (2003).</li></ol>

Here we have presented a detailed cryohistology protocol to co-localize and quantify several biological measures by aligning images from multiple rounds of staining/imaging on a single section. The method outlined using the cryotape is especially useful as it maintains the morphology of difficult to section tissue (e.g., mineralized bone and cartilage). In addition, the sectioned tissue is adhered firmly to the glass slide, allowing for multiple rounds of staining/imaging of the same section; unlike traditional ...

Biological research often requires efficient phenotyping, which is frequently associated with some sort of histological analysis1-3. This need is even more evident with the ambitious projects to disrupt each gene in the mouse genome4. These histological analyses can range from assessing cell morphology and/or anatomical features to mapping expression of specific genes or proteins to individual cells. In fact, one of the fundamental contributions of histology to the field of genomics is the ability to associate a specific molecular signal to a specific region or cell type.
Traditional methods of histology, especially for musculoskeletal tissues, are often time consuming and laborious, requiring sometimes weeks to fix, decalcify, section, stain, and image the specimen then analyze the images via human interpretation. Analyzing multiple molecular signals, whether via immunohistochemistry, in situ hybridization, or special stains, requires multiple sections and even multiple specimens to perform appropriately. In addition, these multiple responses cannot be co-localized to the same cell and sometimes cannot be co-localized to a specific region within a given specimen. As the genomics and epigenomics field moves into the digital age, the histological field must also follow suit to provide efficient, high-throughput, and automated analysis of a variety of molecular signals within a single histological section.
Indeed, there is a demand for improved histological techniques that can associate multiple molecular signals to specific cells within a given specimen. Recently, we have published a new high-throughput cryohistological method for assessing several response measures within a given section from mineralized tissue5-14. The process involves stabilizing the cryosection with frozen cryotape, adhering the taped section rigidly to a microscope slide, and conducting several rounds of staining and imaging on each section. These rounds of images are then aligned manually or via computer automation prior to image analysis (Figure 1). Here, we present detailed protocols of this process and provide examples where these techniques have improved our understanding of different biological processes.

<table border="0" cellpadding="0" cellspacing="0" width="1066">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">10% neutral buffered formalin</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:259px;">HT501128-4L</td>
			<td style="width:359px;">Multiple suppliers available. Toxic. Can be substituted with 4% paraformaldehyde.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S9378</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Sigma Aldrich</td>
			<td>P5368</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomolds</td>
			<td>Fisher Scientific</td>
			<td>Fisherbrand #22-363-554</td>
			<td>Different sizes can be used depending on tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomatrix</td>
			<td>Thermo Scientific</td>
			<td>6769006</td>
			<td>Can be substitituted with other cryomatrices. However, PVA/PEG-based resins have worked best in our hands.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-methyl-butane</td>
			<td>Sigma Aldrich</td>
			<td>M32631</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica Biosystems</td>
			<td>3050s</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Specimen disc</td>
			<td>Leica Biosystems</td>
			<td>14037008587</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat blades</td>
			<td>Thermo Scientific</td>
			<td>3051835</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryotape</td>
			<td>Section Lab</td>
			<td>Cryofilm 2C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roller</td>
			<td>Electron Microscopy Sciences</td>
			<td>62800-46</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic microscope slides</td>
			<td>Electron Microscopy Sciences</td>
			<td>71890-01</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass microscope slides</td>
			<td>Thermo Scientific</td>
			<td>3051</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Norland Optical Adhesive, 61</td>
			<td>Norland Optical</td>
			<td>Norland Optical Adhesive, 61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Black Light</td>
			<td>General Electric</td>
			<td>F15T8-BLB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Sigma Aldrich</td>
			<td>ARK2183</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chitosan</td>
			<td>Sigma Aldrich</td>
			<td>C3646</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InSpeck red microscopheres</td>
			<td>ThermoFisher Scientific</td>
			<td>I-14787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InSpeck green microspheres</td>
			<td>ThermoFisher Scientific</td>
			<td>I-14785</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcein Blue</td>
			<td>Sigma Aldrich</td>
			<td>M1255</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcein</td>
			<td>Sigma Aldrich</td>
			<td>C0875&#160;</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alizarin complexone</td>
			<td>Sigma Aldrich</td>
			<td>A3882&#160;</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Demeclocycline</td>
			<td>Sigma Aldrich</td>
			<td>D6140&#160;</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ELF 97 yellow fluorescent acid phosphatase substrate</td>
			<td>ThermoFisher Scientific</td>
			<td>E-6588</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>62247</td>
			<td>Multiple suppliers available. Can be substituted with Hoechst 33342 or other nuclear dyes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TO-PRO-3 (Cy5 nuclear counterstain)</td>
			<td>ThermoFisher Scientific</td>
			<td>T3605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium Iodide</td>
			<td>ThermoFisher Scientific</td>
			<td>R37108</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>S2889</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium tartrate dibasic dihydrate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T6521</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium nitrite</td>
			<td>Sigma Aldrich</td>
			<td>S2252</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Sigma Aldrich</td>
			<td>15504</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2 hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M9272</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Red TR Salt</td>
			<td>Sigma Aldrich</td>
			<td>F8764</td>
			<td>Multiple suppliers available. Can also be substituted with other substrate kits such as Vector Blue (Vector Laboratories, Cat# SK-5300)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Naphthol AS-MX&#160;</td>
			<td>Sigma Aldrich</td>
			<td>N4875</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>D158550</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toluidine blue O</td>
			<td>Sigma Aldrich</td>
			<td>T3260</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axio Scan.Z1</td>
			<td>Carl Zeiss AG</td>
			<td>Axio Scan.Z1</td>
			<td>Other linear or tiled scanners may also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFP Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">YFP Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Custom yellow (ELF 97) Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>custom; HQ409sp, HQ555/30m, 425dxcr</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRITC Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy5 Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CryoJane Tape Transfer System</td>
			<td>Electron Microscopy Sciences</td>
			<td>62800-10</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CryoJane Tape Windows</td>
			<td>Electron Microscopy Sciences</td>
			<td>62800-72</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CryoJane Adhesive Slides</td>
			<td>Electron Microscopy Sciences</td>
			<td>62800-4X</td>
			<td>Multiple suppliers available.</td>
		</tr>
	</tbody>
</table>

high-throughput, multi-image cryohistology of mineralized tissues

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.

A General Workflow for the High-Throughput, Multi-Image Cryohistology
Figure 1 represents the general workflow used for this technique. It includes several steps from fixation through several rounds of imaging and finally image alignment/analysis. The process can take as little as a week to go from sample fixation through 4 rounds of imaging, which is less time than it takes to decalcify these t...

Watch this Scientific Journal Video about High-Throughput, Multi-Image Cryohistology of Mineralized Tissues at JoVE.com

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

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.

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

The goal of this technology is to provide histological tools that integrate complex model systems in a high-throughput, observer independent, and fully digital manner that is both cost-effective and meaningful to musculoskeletal biologists. Estology, while critical to most biological researchers, is often labor-intensive and non-quantitative. Therefore, there is a need for histological techniques that provide efficient, high-throughput, and automated analysis.With our cryohistology platform, the researcher can associate multiple molecro signals to specific cells within a given specimen by imaging and staining the same slide multiple times in high-throughput manner. Demonstrating the procedure will be Doctor Xi Jiang and Li Chen, research assistants in my laboratory, who have pioneered the development and the execution of these techniques. To begin this procedure, remove the specimen from formalin, dissect away any excess tissue, and transfer to 30%sucrose, made in 1x PBS.Incubate the specimens in sucrose for 12 to 24 hours at four degrees Celsius. Next, fill a cryomold with cryoembedding medium and place the specimens in the mold. Then, align the samples in the mold such that the cutting plain for the region of interest is parallel to the bottom of the mold.After that, place the cryomold onto a piece of dry ice until a thin layer of cryoembedding medium freezes, locking the spicimens into place. Then, fill the remaining volume of the cryomold with cryoembedding medium, while maintaining the cryomold on the dry ice pellet. Following that, place the cryomold in a container containing 2-methylbutane pre-chilled by dry ice.Once the specimens are completely frozen, remove the cryomold and shake off excess 2-methylbutane. Wrap the cryomold in cellophane and store it at minus 20 degrees Celsius, or minus 80 degrees Celsius. In this procedure, transfer the specimen block from the freezer to a cryostat set at minus 20 to minus 25 degrees Celsius.Next, remove the block from the cryomold and trim away excess cryoembedding medium with a razor blade. Then, add some cryoembedding medium onto the specimen disk, and align the block such that the surface of the specimen disk is parallel to the bottom surface of the block. Wait for the cryoembedding medium to freeze.Afterward, place the specimen disk onto the specimen head and adjust the head such that the blade cuts parallel to the surface of the specimen block. Next, cut pieces of cryotape to cover the region of interest and pre-chill them in the cryostat. Afterward, trim the block down to a level within the region of interest, continuing to make adjustments as needed.Once the region of interest has been reached, and a section can be made, brush off any shavings from the surface of the block. Next, remove the non-adherent backing from the cryotape by grasping the tape by the non-sticky silver gold tabs. Then place the tape onto the block with the sticky side down.Apply pressure to the cryotape using the roller, and make a section using either the automatic motor or manual cutting wheel of the cryostat. Following this, place the section tissue side up on a plastic microscope slide within the cryostat. Remove the plastic slide from the cryostat and allow the cryoembedding medium to melt, such that the section adheres to the slide's surface.Repeat sectioning for the serial sections or other regions of interest. Once finished, place the sections in a slide box and store them at four, minus 20, or minus 80 degrees Celsius, depending on the length of storage required and the downstream response measures. To perform the UV curable adhesive method, first label the microscope slide.Apply a drop of UV activated optical adhesive to two glass slides, and use the edge of a slide to spread a thin layer of adhesive across the surface of the glass slides. Then, cut off the silver gold tab and lay the taped section tissue side up, on the adhesive layer of the first slide. Apply the section in a rolling motion from one edge to the other in order to minimize the formation of entrapped bubbles underneath the tape.Afterward, remove the taped section from the first slide, then place it on the adhesive layer of the second slide, and take care to avoid entrapment of bubbles. Once the appropriate number of sections for the given experiment is placed on each slide, wipe off the excess adhesive from the surrounding areas. Next, inspect the sections closely for bubbles or fibers underneath the tape.Then place the microscope slides under the UV black light for five to ten minutes to cross-link the adhesive layer. In this step, prepare the reference marker solution by dissolving 50 microliters of green, and 50 microliters of red microspheres in 100 microliters of water. Store the solution in the dark at four degrees Celsius.Then dip a pipette tip into the microsphere solution, and dab the microsphere solution onto each section, adjacent to the region of interest. Next, dry the slides at room temperature and protect them from light by covering them with foil for 30 minutes, if processing the slides on that day. If not, place the slides in a slide box at four degrees Celsius.After staining the sections, mount the cover slips on the slides using acquiesce mounting medium. To perform imaging, load the slides into the microscope trays and insert the trays into the tray stack of the slide scanning microscope. Then, click on the profile list in the microscope software to load a profile for each slide with the appropriate exposure time for each flurophore.The, click on the Start Preview Scan button to take a preview image of each slide. The software will apply a file name automatically by reading the bar codes placed on the slide labels. Alternatively, a file name can be manually assigned to each slide.Set the regions of interest by entering the tissue detection wizard, drawing each region using the draw tool, making any necessary adjustments, and saving them for subsequent rounds of imaging. Once each slide is set up, start the scan process by clicking the Start Scan button. Following each round of imaging, submerge the slides in a coplin jar filled with one 1x PBS until the cover slips fall off from the slides, before the next staining step.After all of the imaging rounds have been completed, export the images from each individual channel as TIF or JPG files for image assembly and analysis. In this figure, a section from a three month old mouse femur was stained with calcein blue and imaged for accumulated mineral, seen in white, and mineralization labels, seen in green and red in the first round of imaging. Slides were then imaged for trap activity, seen in yellow in the second round, and AP activity, seen in red in the third round of imaging.Finally, the slides were stained with toluidine blue in the fourth round. The reference markers were imaged during every imaging round including the chromogenic round, and were aligned using the custom software. Once mastered, this technique can be done in less time than it takes to decalcify a typical specimen.After watching this video, you should have a good understanding of how to produce high quality high content images of mineralized tissues, which we hope can be more broadly used by the musculoskeletal community.

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Research

JoVE Journal

Biology

15.7K 조회수.  University of Connecticut Health Center. The goal of this technology is to provide histological tools that integrate complex model systems in a high-throughput, observer independent, and fully digital manner that is both cost-effective and meaningful to musculoskeletal biologists. Estology, while critical to most biological researchers, is often labor-intensive and non-quantitative. Therefore, there is a need for histological techniques that provide efficient, high-throughput, and automated analysis.With our cryohistology pla...