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

<ol>
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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>

The authors declare that they have no competing financial interests.

Aquí hemos presentado un protocolo detallado cryohistology co-localizar y cuantificar varias medidas biológicas mediante la alineación de imágenes de varias rondas de manchas / formación de imágenes en una sola sección. El método descrito mediante el cryotape es especialmente útil, ya que mantiene la morfología de difícil sección de tejido (por ejemplo, hueso mineralizado y el cartílago). Además, el tejido seccionado se adhiere firmemente a la lámina de vidrio, lo que permite múltiples rondas de...

La investigación biológica requiere a menudo fenotipificación eficiente, lo que con frecuencia se asocia con algún tipo de análisis histológico 1-3. Esta necesidad es aún más evidente con los ambiciosos proyectos para interrumpir cada gen en el genoma del ratón 4. Estos análisis histológicos pueden variar desde la evaluación de la morfología celular y / o características anatómicas a la expresión de mapeo de genes específicos o proteínas a las células individuales. De hecho, una de las contribuciones fundamentales de la histología en el campo de la genómica es la posibilidad de asociar una señal molecular específica a una región o célula tipo específico. Los métodos tradicionales de la histología, especialmente para los tejidos musculoesqueléticos, son a menudo mucho tiempo y laborioso, que requiere a veces semanas para arreglar, descalcificación, sección, mancha, e imagen de la muestra a continuación, analizar las imágenes a través de la interpretación humana. El análisis de señales moleculares múltiples, ya sea a través de inmunohistoquímica, en hybridizat situiones, o tinciones especiales, requiere múltiples secciones e incluso múltiples muestras para llevar a cabo adecuadamente. Además, estas respuestas múltiples no pueden ser co-localizan en la misma célula y a veces puede no ser co-localizada a una región específica dentro de una determinada muestra. Como el campo de la genómica y la epigenomics mueve a la era digital, el campo histológico también debe seguir el juego para proporcionar eficiente, de alto rendimiento, y el análisis automatizado de una variedad de señales moleculares dentro de una sola sección histológica. De hecho, existe una demanda de mejora de las técnicas histológicas que se pueden asociar múltiples señales moleculares a células específicas dentro de una determinada muestra. Recientemente, se ha publicado un nuevo método cryohistological de alto rendimiento para la evaluación de varias medidas de respuesta dentro de una sección dada de tejido mineralizado 5-14. El proceso consiste en la estabilización de la sección criogénica con cryotape congelado, adherir la sección de cinta adhesiva de forma rígida a un portaobjetos de microscopio, yla realización de varias rondas de tinción y de imagen en cada sección. Estas rondas de imágenes son entonces alineados manualmente o por medio de la automatización de equipo antes de análisis de imagen (Figura 1). A continuación, presentamos los protocolos detallados de este proceso y proporcionar ejemplos en los que estas técnicas han mejorado nuestra comprensión de los diferentes procesos biológicos.

<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.

Un flujo de trabajo general para el de alto rendimiento, de múltiples imágenes Cryohistology La figura 1 representa el flujo de trabajo general usado para esta técnica. Incluye varios pasos de fijación a través de varias rondas de formación de imágenes y, finalmente, la imagen de alineación / análisis. El proceso puede tomar tan poco como una semana para ir de la fijación de la muest...

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.

De alto rendimiento, de múltiples imágenes Cryohistology de tejidos mineralizados

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

high-throughput-multi-image-cryohistology-of-mineralized-tissues

Research

JoVE Journal

Biology

15.8K vistas.  University of Connecticut Health Center. 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. 

Video: High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

La levadura de alto rendimiento de la pantalla sobreexpresión plásmido

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Investigación

Biología

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 a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

De alto rendimiento de sacarificación de ensayo para materiales lignocelulósicos

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 Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

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 biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

De alto rendimiento de purificación de afinidad de proteínas marcadas con recombinantes

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 treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

High Throughput microinyecciones de Sea Urchin cigotos

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 viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

De Alto Rendimiento La titulación de luciferasa que expresan virus recombinantes

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 purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperación de adultos de pez cebra Corazones para aplicaciones de alto rendimiento

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 focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

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 signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificación de los pares quinasa-sustrato Usando selección de alto rendimiento


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

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput<em&gt; Danio rerio</em&gt; Ensayo gasto energético

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