Name: simple detection of primary cilia by immunofluorescence
Uploaded: 2020-05-15T14:00:00
Description: Watch this Scientific Journal Video about Simple Detection of Primary Cilia by Immunofluorescence at JoVE.com

Este trabajo fue apoyado por el Ministerio de Defensa de la República Checa - Plan de desarrollo de organización a largo plazo Aspectos médicos de las armas de destrucción masiva de la Facultad de Ciencias de la Salud Militar, Universidad de Defensa; ministerio de Educación, Juventud y Deporte, República Checa (Proyecto de Investigación Específico No: SV/ FVZ201703) y PROGRES Q40/06. Gracias también a Daniel Díaz por su amable ayuda en la revisión del idioma inglés.

1. Preparación de medios de cultivo, soluciones y platos
<ol><li>Autoclave los cubreobjetos (22 x 22 mm). Prepare 6 placas de pozo. Descongelar el suero bovino fetal (FBS) y la penicilina/estreptomicina antibiótica y calentar el cultivo a temperatura media a ambiente (RT). Usar trypsin-EDTA (0,25%) y 1x PBS (solución salina tamponada de fosfato con calcio y magnesio) para pasar las células.</li>
	<li>Preparar paraformaldehído fresco 4% (PFA) en dH2O (800 mg de PFA en 20 ml de dH2O). El PFA de...

<ol>
	<li>Alieva, I. B., Vorobjev, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+primary+cilia:+a+sensory+part+of+centrosomal+complex+in+tissue+cells,+but+a+"sleeping+beauty"+in+cultured+cells.">Vertebrate primary cilia: a sensory part of centrosomal complex in tissue cells, but a "sleeping beauty" in cultured cells.</a> Cell Biology International. 28 (2), 139-150 (2004).</li><li>Sloboda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primary cilia. , (2009).</li><li>Sharma, N., Kosan, Z. A., Stallworth, J. E., Berbari, N. F., Yoder, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+levels+of+cytosolic+tubulin+regulate+ciliary+length+control.">Soluble levels of cytosolic tubulin regulate ciliary length control.</a> Molecular Biology of the Cell. 22 (6), 806-816 (2011).</li><li>Filipov&#225;, 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=Ionizing+radiation+increases+primary+cilia+incidence+and+induces+multiciliation+in+C2C12+myoblasts.">Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts.</a> Cell Biology International. 39 (8), 943-953 (2015).</li><li>Filipov&#225;, 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=The+toxic+effect+of+cytostatics+on+primary+cilia+frequency+and+multiciliation.">The toxic effect of cytostatics on primary cilia frequency and multiciliation.</a> Journal of Cellular and Molecular Medicine. 23 (8), 5728-5736 (2019).</li><li>Gadadhar, S., 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=Tubulin+glycylation+controls+primary+cilia+length.">Tubulin glycylation controls primary cilia length.</a> The Journal of Cell Biology. 216 (9), 2701-2713 (2017).</li><li>Shamloo, K., 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=Chronic+Hypobaric+Hypoxia+Modulates+Primary+Cilia+Differently+in+Adult+and+Fetal+Ovine+Kidneys.">Chronic Hypobaric Hypoxia Modulates Primary Cilia Differently in Adult and Fetal Ovine Kidneys.</a> Frontiers in Physiology. 8, 677 (2017).</li><li>Malone, A. M. 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=Primary+cilia+mediate+mechanosensing+in+bone+cells+by+a+calcium-independent+mechanism.">Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (33), 13325-13330 (2007).</li><li>Luesma, M. J., 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=Enteric+neurons+show+a+primary+cilium.">Enteric neurons show a primary cilium.</a> Journal of Cellular and Molecular Medicine. 17 (1), 147-153 (2013).</li><li>Pugacheva, E. N., Jablonski, S. A., Hartman, T. R., Henske, E. P., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEF1-dependent+Aurora+A+activation+induces+disassembly+of+the+primary+cilium.">HEF1-dependent Aurora A activation induces disassembly of the primary cilium.</a> Cell. 129 (7), 1351-1363 (2007).</li><li>Li, 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=Ciliary+transition+zone+activation+of+phosphorylated+Tctex-1+controls+ciliary+resorption,+S-phase+entry+and+fate+of+neural+progenitors.">Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors.</a> Nature Cell Biology. 13 (4), 402-411 (2011).</li><li>Spalluto, C., Wilson, D. I., Hearn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+reciliation+of+RPE1+cells+in+late+G1+phase,+and+ciliary+localisation+of+cyclin+B1.">Evidence for reciliation of RPE1 cells in late G1 phase, and ciliary localisation of cyclin B1.</a> FEBS Open Bio. 3, 334-340 (2013).</li><li>Malicki, J. J., Johnson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cilium:+Cellular+Antenna+and+Central+Processing+Unit.">The Cilium: Cellular Antenna and Central Processing Unit.</a> Trends in Cell Biology. 27 (2), 126-140 (2017).</li><li>Morleo, M., Franco, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Autophagy-Cilia+Axis:+An+Intricate+Relationship.">The Autophagy-Cilia Axis: An Intricate Relationship.</a> Cells. 8 (8), 905 (2019).</li><li>Ott, C., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+live+primary+cilia+dynamics+using+fluorescence+microscopy.">Visualization of live primary cilia dynamics using fluorescence microscopy.</a> Current Protocols in Cell Biology. , (2012).</li><li>Sun, S., Fisher, R. L., Bowser, S. S., Pentecost, B. T., Sui, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+architecture+of+epithelial+primary+cilia.">Three-dimensional architecture of epithelial primary cilia.</a> Proceedings of the National Academy of Sciences. 116 (19), 9370-9379 (2019).</li><li>Lauring, M. C., 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=New+software+for+automated+cilia+detection+in+cells+(ACDC).">New software for automated cilia detection in cells (ACDC).</a> Cilia. 8 (1), 1 (2019).</li><li>Conroy, P. C., 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=C-NAP1+and+rootletin+restrain+DNA+damage-induced+centriole+splitting+and+facilitate+ciliogenesis.">C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.</a> Cell Cycle. 11 (20), 3769-3778 (2012).</li><li>Kim, J. H., 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=Genome-wide+screen+identifies+novel+machineries+required+for+both+ciliogenesis+and+cell+cycle+arrest+upon+serum+starvation.">Genome-wide screen identifies novel machineries required for both ciliogenesis and cell cycle arrest upon serum starvation.</a> Biochimica et Biophysica Acta. 1863 (6), 1307-1318 (2016).</li><li>Yuan, K., 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=Primary+cilia+are+decreased+in+breast+cancer:+analysis+of+a+collection+of+human+breast+cancer+cell+lines+and+tissues.">Primary cilia are decreased in breast cancer: analysis of a collection of human breast cancer cell lines and tissues.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 58 (10), 857-870 (2010).</li><li>Smith, Q., 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=Differential+HDAC6+Activity+Modulates+Ciliogenesis+and+Subsequent+Mechanosensing+of+Endothelial+Cells+Derived+from+Pluripotent+Stem+Cells.">Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells.</a> Cell Reports. 24 (4), 895-908 (2018).</li><li>Mirvis, M., Siemers, K. A., Nelson, W. J., Stearns, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cilium+loss+in+mammalian+cells+occurs+predominantly+by+whole-cilium+shedding.">Primary cilium loss in mammalian cells occurs predominantly by whole-cilium shedding.</a> PLOS Biology. 17 (7), 3000381 (2019).</li><li>Hua, K., Ferland, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+methods+can+differentially+affect+ciliary+protein+immunolabeling.">Fixation methods can differentially affect ciliary protein immunolabeling.</a> Cilia. 6 (1), 5 (2017).</li><li>Lim, Y. C., McGlashan, S. R., Cooling, M. T., Long, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+detection+of+primary+cilia+in+endothelial+cell+models.">Culture and detection of primary cilia in endothelial cell models.</a> Cilia. 4, 11 (2015).</li><li>DiDonato, D., Brasaemle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+methods+for+the+study+of+lipid+droplets+by+immunofluorescence+microscopy.">Fixation methods for the study of lipid droplets by immunofluorescence microscopy.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 51 (6), 773-780 (2003).</li></ol>

Varios autores han descrito diversos métodos para la detección de cilios primarios, a veces también describiendo varios métodos de fijación que pueden afectar a su detección6,,20,,21,,22. A pesar de todo, es difícil encontrar un protocolo completo y directo para la detección. La disponibilidad de tal método sería sin duda de gran ayuda para el estudio de la investigación de cilios pr...

Los cilios primarios son estructuras sensoriales, solitarias, ligadas a la membrana y no móviles asociadas con el centriole maternés de la célula. Los cilios primarios se encuentran en la mayoría de las células vertebradas con la excepción de los glóbulos rojos, adipocitos1y hepatocitos2. Los cilios primarios se forman como un axonemo alargado compuesto por microtúbulos, cuyo componente principal es la tubulina. El axonemo crece a partir del cuerpo basal, que está estructurado a partir de la tubulina. La longitud de los cilios primarios varía entre 2–10 m; sin embargo, sus dimensiones pueden cambiar durante la glicación, inanición, hipoxia, estrés citotóxico, o después de la exposición a la radiación ionizante3,4,5,6,7. Por lo general, las células tienen sólo un cilium primario, que participa en la morfogénesis y las vías de señalización celular importantes para la proliferación celular y la diferenciación8,9.
Los cilios primarios se regulan dinámicamente durante la progresión del ciclo celular, específicamente durante las fases G0/G1, y se reabsorben antes de entrar en la mitosis en un proceso asociado con la deacetilación de la tubulina mediada por HDAC6 (histona deacetilasa 6)10. El momento exacto de la resorción primaria de los cilios depende del tipo de célula y de la expresión de genes directamente implicados en este proceso, como Aurora A, Plk1, TcTex-111,12,,13. Dependiendo del tipo de célula, los cilios primarios expresan diferentes tipos de receptores, canales iónicos y vías de señalización activas. Estos incluyen los receptores de señalización más importantes que afectan a la proliferación y supervivencia, EGFR, PDGFR, y FGFR. También se incluyen algunas de las vías de señalización que pueden afectar la función de uno o más órganos, incluyendo erizo, muesca, y Wnt. Gracias a estos receptores y vías de señalización, los cilios primarios también realizan una función quimiosensorial. Esta función permite a los cilios primarios detectar ligandos específicos para la muesca, hormonas, y sustancias biológicamente activas como la serotonina o somatostatina. Otras funciones específicas exhibidas por cilios primarios de diferentes longitudes incluyen la reacción a los cambios de temperatura, gravedad y osmolalidad14.
Los cilios primarios se pueden visualizar a través de diversos métodos, como la visualización en vivo, la microscopía electrónica de transmisión, la imagen 3D o por software para la detección automática de cilios primarios5,,15,,16,,17. Sin embargo, estos métodos son métodos de investigación altamente especializados y continuos para los métodos básicos, rápidos y fáciles para tinr a los cilios primarios en cada etapa de la investigación. Descrito es un método fácil y útil para la detección de cilios primarios en células cultivadas.

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			<td height="21" style="height:21px;width:384px;">6-well plate</td>
			<td style="width:217px;">TPP</td>
			<td style="width:119px;">92406</td>
			<td style="width:581px;">Dimensions 128x86x22 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Alexa Fluor488</td>
			<td style="width:217px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">111-546-047</td>
			<td>AffiniPure F(ab&#39;)&#8322; Fragment Goat Anti-Rabbit IgG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Anti-Tubulin &#947;</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">T5192</td>
			<td>Polyclonal Rabbit anti-Mouse IgG2a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">C2C12</td>
			<td style="width:217px;">ATCC</td>
			<td style="width:119px;">CRL-1772</td>
			<td>Myoblast (mouse)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Cy3</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">C2181</td>
			<td>Anti-Mouse IgG (whole molecule) F(ab&#8242;)2 fragment&#8211;Cy3 antibody produced in sheep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Dapi (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Dulbecco&#180;s Modified Eagle&#180;s medium</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">11960044</td>
			<td>High glucose, No glutamine, Gibco</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:384px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td>With MgCl2 and CaCl2, Sterile-filtered, Suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Fetal Bovine Serum</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">16000044</td>
			<td>Sterile-Filtered, Gibco</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:384px;">L-Glutamine</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">MEF</td>
			<td style="width:217px;">ATCC</td>
			<td style="width:119px;">SCRC-1039</td>
			<td>Mouse embryonic fibroblast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Monoclonal Anti-Acetylated Tubulin</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7451</td>
			<td>Monoclonal Anti-Acetylated Tubulin antibody produced in mouse</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:384px;">NHLF</td>
			<td style="width:217px;">Lonza</td>
			<td style="width:119px;">CC-2512</td>
			<td>Primary lung fibroblasts (human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Normal Goat Serum</td>
			<td style="width:217px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Paraformaldehyde</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">158127-500G</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Penicillin-Streptomycin</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">P0781</td>
			<td>10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, Sterile-Filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">ProLong Diamond Antifade Mountant</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">P36961</td>
			<td></td>
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			<td height="63" style="height:63px;width:384px;">Skin fibroblasts</td>
			<td style="width:217px;">Kindly gifted from Charles University, Faculty of Medicine in Hradec Kr&#225;lov&#233;.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Square Cover Slips</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">22X22-1.5</td>
			<td>Borosilicate glass, 22x22mm, Square</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Triton X-100</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">11332481001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">25200072</td>
			<td>Sterile-Filtered, Gibco</td>
		</tr>
	</tbody>
</table>

simple detection of primary cilia by immunofluorescence

Los cilios primarios se regulan dinámicamente durante la progresión del ciclo celular, específicamente durante las fases G0/G1 del ciclo celular, siendo reabsorbidos antes de la mitosis. Los cilios primarios se pueden visualizar con métodos altamente sofisticados, incluyendo microscopía electrónica de transmisión, imágenes 3D o el uso de software para la detección automática de cilios primarios. Sin embargo, la tinción inmunofluorescente de los cilios primarios es necesaria para realizar estos métodos. Esta publicación describe un protocolo para la fácil detección de cilios primarios in vitro mediante la tinción de la tubulina alfa acetilada (axoneme) y la tubulina gamma (cuerpo basal). Este protocolo de tinción inmunofluorescente es relativamente simple y da como resultado imágenes de alta calidad. El presente protocolo describe cómo cuatro líneas celulares (C2C12, MEF, NHLF y fibroblastos cutáneos) que expresaban cilios primarios fueron fijadas, inmunotendadas e imágenes con un microscopio fluorescente o confocal.

La tinción inmunofluorescente de los cilios primarios es un procedimiento relativamente simple que da como resultado imágenes de alta calidad. En estos experimentos, los fibroblastos que expresaban cilios primarios fueron fijos, inmunotuvistedos e imágenes en un microscopio fluorescente o confocal siguiendo el protocolo descrito anteriormente. El cilium primario se detectó utilizando la tubulina acetilada y la tubulina. La evaluación de los cilios primarios se puede realizar en varios niveles y cualquier cambio en e...

Watch this Scientific Journal Video about Simple Detection of Primary Cilia by Immunofluorescence at JoVE.com

Simple Detection of Primary Cilia by Immunofluorescence

Los cilios primarios son estructuras extracelulares asociadas con el centriolo. La detección de cilios primarios por tinción inmunofluorescente es un procedimiento relativamente simple que da como resultado imágenes de muy alta calidad. En este protocolo, los fibroblastos que expresaban cilios primarios fueron fijos, inmunosutenidos e imágenes en un microscopio fluorescente o confocal.

Detección simple de cilios primarios por inmunofluorescencia

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

Este protocolo permite la detección rápida y fácil de cilios primarios en células cultivadas, dentro de una variedad de esfuerzos de investigación. El método podría ser utilizado en el trastorno que afecta a los cuerpos basales de los cilios primarios. Este procedimiento también se puede aplicar para la tinción de cilios primarios en tejido incrustado de parafina o para otros experimentos donde se necesita fluorescencia.Comience por autoclaving 22 por 22 milímetros de cubierta, y la preparación de materiales para el experimento. Descongelar FBS y penicilina estreptomicina, y calentar el cultivo de temperatura media a ambiente. Pasaje de las células con trippsina EDTA y PBS.Preparar 4%paraformaldehído fresco, medio de cultivo y solución de gelatina al 1%, según las instrucciones del manuscrito. A continuación, limpie la campana de flujo laminar con 70% de etanol y coloque todos los materiales necesarios en el interior. El paraformaldehído es extremadamente peligroso.El EPP adecuado tiene que ser usado en todo momento, y sólo debe ser manejado en una capucha química. Después de cultivar las células deseadas al 70%confluencia, retírelas de la incubadora y colóquelas en la campana de flujo laminar. Retire el medio de cultivo y enjuague las células dos veces con PBS.Añadir alrededor de dos mililitros de 0.25%trypsin EDTA al matraz de cultivo, e incubarlo a 37 grados Centígrados durante cinco minutos. Revise las células periódicamente en el microscopio invertido para monitorear el desprendimiento. Cuando las células se han separado suavemente las resuspend en 10 mililitros de medio de cultivo, pipeteando cuidadosamente para crear una sola suspensión de celda.Transfiera la suspensión celular a un tubo cónico de 50 mililitros y centrifúquela a 200 veces G'for cinco minutos. Decantar el sobrenadante luego añadir 10 mililitros de medio de cultivo, y resuspender suavemente el pellet. Tome 20 microlitros de la suspensión celular y mezcle con el azul de tripano en una relación de volumen uno a uno.A continuación, cuente las células utilizando hemocitometría estándar. Coloque un resbalón de cubierta dentro de cada pozo de una placa de seis pozos, y cubra el resbalón de la cubierta con dos mililitros de la solución de gelatina, lo que ayudará a que las células unidas a la cubierta se resbalen. Retire la gelatina y deje que la placa se seque al aire durante unos minutos.Semillas 100.000 fibroblastos en cada pozo y añadir dos mililitros de medio de cultivo. Luego incubar las células durante 24 horas a 37 grados Centígrados, cinco por ciento de dióxido de carbono y 90% de humedad relativa. Después de la incubación tratar las células para inducir la ciliación.Caliente el 4%paraformaldehído a temperatura ambiente, y prepare una pipeta de pastos, ABS, un contenedor de residuos, tubos cónicos de 15 mililitros, micro pipetas y puntas. Tome las células de la incubadora y colóquelas en el banco del laboratorio. Retire los medios de cada pozo, dejando el resbalón de la cubierta.Lave suavemente las células tres veces con dos mililitros de PBS. Luego agregue dos mililitros del 4%PFA en cada pozo para fijar las celdas. Incubar la placa durante 10 minutos a temperatura ambiente, luego retirar el PFA y repetir los lavados con PBS.Añadir dos mililitros de 0.5%Tritón X-100 a cada pozo, e incubar la placa durante 15 minutos. A continuación, lave los pozos cuatro veces con PBS. Para hacer la solución de bloqueo, descongele un suero de cabra durante cinco minutos y diluya en PBS en una proporción de uno a 20.Añadir 150 microlitros de esta solución a cada resbalón de cubierta e incubar la placa durante 20 minutos. Descongelar los anticuerpos primarios durante cinco minutos, y diluirlos por separado en PBS, como se describe en el manuscrito de texto. Retire la solución de bloqueo de los resbalones de la cubierta y añada 150 microlitros de ambas soluciones de anticuerpos.A continuación, incubar la placa durante 60 minutos a temperatura ambiente. Retire los anticuerpos primarios y lave suavemente los resbalones de la cubierta tres veces con dos mililitros de PBS. Diluir Cy3 Sheep Anti-Mouse y Alexa fluor 488 Goat anti-Rabbit en PBS, a una proporción de uno a 300, y añadir 150 microlitros de ambas soluciones de anticuerpos secundarios al resbalón de la cubierta.Preparar una solución DAPI en funcionamiento diluyendo 10 microlitros del stock en 50 mililitros de PBS, y añadir dos mililitros de esta dilución a los resbalones de la cubierta. Incubar el plato durante cinco minutos en la oscuridad, a temperatura ambiente. Antes de colocar los resbalones de la cubierta en los portaobjetos, prepare dos agujas, pinzas y medios de montaje, y etiquete todas las diapositivas.Retire la solución DAPI de los pozos y lávela tres veces con PBS. Coloque una gota de medios de montaje en cada diapositiva. Utilice la aguja para levantar suavemente el resbalón de la cubierta desde la parte inferior del pozo.A continuación, voltéelo y colóquelo suavemente sobre la gota de los medios de montaje con las pinzas. Retire con cuidado las burbujas. Proteja los portaobjetos de la luz y guárdelos durante la noche a cuatro grados centígrados.Al día siguiente utilice un microscopio fluorescente o confocal con un alto aumento para visualizar a los cilios primarios. Este protocolo se utilizó para fijar inmunosu mancha, e imágenes de varias líneas celulares que expresan cilios primarios. Cuando los mioblastos de ratón fueron expuestos a la radiación ionizante a varias dosis, se encontró que dosis más altas inducen la aparición de múltiples cilios primarios.Mientras que dosis bajas resultaron en la aparición de cilios primarios individuales. Del mismo modo, cuando los fibroblastos pulmonares humanos se irradiaban a una dosis baja, los cilios primarios únicos se detectaron por inmunofluorescencia. Se observó un aumento de los cilios primarios en fibroblastos embrionarios de ratón que murieron de hambre, lo que demuestra que el estrés metabólico afecta a los incidentes de los cilios primarios.Cuando los fibroblastos cutáneos fueron tratados con 120 doxorubicina nanomolares, expresaron un único cilium primario. Dosis más altas inducen la aparición de múltiples cilios primarios. Los fibroblastos tratados con 1,25 Taxol nanomolar también expresaron un único cilium primario.Pero no se detectaron varios cilios después del tratamiento con dosis más altas. Al abrir este protocolo, asegúrese de seguir todas las regulaciones de EPI. Tenga en cuenta las necesidades de cultivo de su línea celular de elección y elija sus anticuerpos cuidadosamente.Este procedimiento no puede ser seguido directamente por otros procedimientos, ya que las células son irrecuperables. En su lugar, deben programarse experimentos paralelos.

simple-detection-of-primary-cilia-by-immunofluorescence

Research

JoVE Journal

Biology

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

Detección por inmunofluorescencia de dos análogos de la timidina (CldU y UDI) en los tejidos de Primaria

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

Investigación

Biología

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

La detección de metaloproteinasas de la matriz funcional por Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

La detección de modificaciones post-traduccionales de los nativos intactos nucleosomas por ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

La detección de ARN viral por fluorescencia<em&gt; In situ</em&gt; La hibridación (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

El análisis de la función génica y visualización de los cilios-generó un flujo de líquido en la vesícula Kupffer

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

La detección de vivo<em&gt; Escherichia coli</em&gt; O157: H7 células por PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Análisis de la apoptosis en embriones de pez cebra en Todo el montaje de inmunofluorescencia para detectar la caspasa 3 activada

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

La detección de activados por ligando G receptor acoplado a proteína internalización por microscopía confocal

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...