Name: high-throughput, multi-image cryohistology of mineralized tissues
Uploaded: 2016-09-14T13:00:00
Description: Watch this Scientific Journal Video about High-Throughput, Multi-Image Cryohistology of Mineralized Tissues at JoVE.com

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 ...

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High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

High Throughput Danio Rerio Energy Expenditure Assay

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...