Name: immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos
Uploaded: 2016-02-19T14:00:00
Description: Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

עבודה זו נתמכה על ידי מענק הקרן הלאומית למדע IOS-0,817,658.

הנהלים דג הזברה בפרוטוקול זה אושרו על ידי ועדת טיפול בבעלי חיים מוסדיים השתמש (IACUC) באוניברסיטת וירג&#39;יניה. 1. הכנת ריאגנטים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 4% PFA / PBS. לשקול 8 גרם של paraformaldehyde (PFA) ב למכסה המנוע קטר. עו?...

<ol>
	<li>Webb, S. E., Miller, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling+during+embryonic+development.">Calcium signalling during embryonic development.</a> Nat Rev Mol Cell Biol. 4, 539-551 (2003).</li><li>Webb, S. E., Miller, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++signalling+and+early+embryonic+patterning+during+zebrafish+development.">Ca2+ signalling and early embryonic patterning during zebrafish development.</a> Clin Exp Pharmacol Physiol. 34, 897-904 (2007).</li><li>Whitaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+at+fertilization+and+in+early+development.">Calcium at fertilization and in early development.</a> Physiol. Rev. 86, 25-88 (2006).</li><li>Yuen, M. 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=Characterization+of+Ca(2+)+signaling+in+the+external+yolk+syncytial+layer+during+the+late+blastula+and+early+gastrula+periods+of+zebrafish+development.">Characterization of Ca(2+) signaling in the external yolk syncytial layer during the late blastula and early gastrula periods of zebrafish development.</a> Biochim Biophys Acta. 1833, 1641-1656 (2013).</li><li>Tombes, R. M., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+free+calcium+and+mitosis+in+mammalian+cells:+anaphase+onset+is+calcium+modulated,+but+is+not+triggered+by+a+brief+transient.">Intracellular free calcium and mitosis in mammalian cells: anaphase onset is calcium modulated, but is not triggered by a brief transient.</a> J. Cell Biol. 109, 627-636 (1989).</li><li>Hudmon, A., Schulman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Ca2+/Calmodulin-Dependent+Protein+Kinase+II:+The+Role+of+Structure+and+Autoregulation+in+Cellular+Function.">Neuronal Ca2+/Calmodulin-Dependent Protein Kinase II: The Role of Structure and Autoregulation in Cellular Function.</a> Annu. Rev. Biochem. 71, 473-510 (2002).</li><li>Tombes, R. M., Faison, M. O., Turbeville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+Evolution+of+Multifunctional+Ca2+/CaM-dependent+Protein+Kinase+(CaMK-II).">Organization and Evolution of Multifunctional Ca2+/CaM-dependent Protein Kinase (CaMK-II).</a> Gene. 322, 17-31 (2003).</li><li>Braun, A. P., Schulman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Multifunctional+Calcium/Calmodulin-Dependent+Protein+Kinase:+From+Form+to+Function.">The Multifunctional Calcium/Calmodulin-Dependent Protein Kinase: From Form to Function.</a> Annu. Rev. Physiol. 57, 417-445 (1995).</li><li>Rich, R. C., Schulman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-directed+function+of+calmodulin+in+autophosphorylation+of+Ca2+/calmodulin-dependent+protein+kinase+II.">Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.</a> J Biol Chem. 273, 28424-28429 (1998).</li><li>Rothschild, S. 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=Tbx5-mediated+expression+of+Ca2+/calmodulin-dependent+protein+kinase+II+is+necessary+for+zebrafish+cardiac+and+pectoral+fin+morphogenesis.">Tbx5-mediated expression of Ca2+/calmodulin-dependent protein kinase II is necessary for zebrafish cardiac and pectoral fin morphogenesis.</a> Dev Biol. 330, 175-184 (2009).</li><li>Rothschild, S. C., Lister, J. A., Tombes, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+CaMK-II+genes+during+early+zebrafish+embryogenesis.">Differential expression of CaMK-II genes during early zebrafish embryogenesis.</a> Dev Dyn. 236, 295-305 (2007).</li><li>Francescatto, L., Rothschild, S. C., Myers, A. L., Tombes, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+activation+of+membrane+targeted+CaMK-II+in+the+zebrafish+Kupffer's+vesicle+is+required+for+left-right+asymmetry.">The activation of membrane targeted CaMK-II in the zebrafish Kupffer's vesicle is required for left-right asymmetry.</a> Development. 137, 2753-2762 (2010).</li><li>Rothschild, S. C., Francescatto, L., Drummond, I. A., Tombes, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMK-II+is+a+PKD2+target+that+promotes+pronephric+kidney+development+and+stabilizes+cilia.">CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia.</a> Development. 138, 3387-3397 (2011).</li><li>Rothschild, S. 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=CaMK-II+activation+is+essential+for+zebrafish+inner+ear+development+and+acts+through+Delta-Notch+signaling.">CaMK-II activation is essential for zebrafish inner ear development and acts through Delta-Notch signaling.</a> Dev Biol. 381, 179-188 (2013).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. , e1113 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. , e1115 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A guide for the laboratory use of zebrafish (Brachydanio rerio). , (1993).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Obara, 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=Polycystin-2+immunolocalization+and+function+in+zebrafish.">Polycystin-2 immunolocalization and function in zebrafish.</a> J Am Soc Nephrol. 17, 2706-2718 (2006).</li><li>Chitramuthu, B. P., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+whole+mount+in+situ+hybridization+within+zebrafish+embryos+to+study+gene+expression+and+function.">High resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function.</a> J Vis Exp. , e50644 (2013).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+in+situ+hybridization+to+whole-mount+zebrafish+embryos.">High-resolution in situ hybridization to whole-mount zebrafish embryos.</a> Nature. 3, 59-69 (2008).</li><li>Harris, P., Osborn, M., Weber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+tubulin-containing+structures+in+the+egg+of+the+sea+urchin+Strongylocentrotus+purpuratus+from+fertilization+through+first+cleavage.">Distribution of tubulin-containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage.</a> J Cell Biol. 84, 668-679 (1980).</li></ol>

השיטה PFA / מתנול פותחה במעבדה זה במטרה העיקרית של אופטימיזציה של immunolocalization של epitope phospho-T 287 -CaMK-II במהלך ההתפתחות דג הזברה. שיטה זו מקומי בהצלחה P-CaMK-II במהלך היווצרות של מספר איברים ריסי כולל KV דג הזברה, 12 האוזן הפנימית 14 וכליות. 13 במיוחד בשלב KV, טכני?...

The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals. 
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred. 
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer's Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer's Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">1-phenyl-2-thiourea (PTU)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P-7629</td>
			<td style="width:347px;">0.12% Stock solution. Dilute 1:40 in system water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 anti-mouse IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11001</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 anti-rabbit IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11008</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa488 phalloidin</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A12379</td>
			<td>Preferentially binds to F-actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa568 anti-mouse IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11004</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Alexa568 anti-rabbit IgG</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">A11011</td>
			<td style="width:347px;">Goat polyclonal, use at 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-acetylated a-tubulin</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">T7451</td>
			<td style="width:347px;">Mouse monoclonal, use at 1:500</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:247px;">anti-phospho-T287 CaMK-II</td>
			<td style="width:175px;">EMD Millipore</td>
			<td style="width:119px;">06-881</td>
			<td style="width:347px;">Rabbit polyclonal, use at 1:20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-total CaMK-II</td>
			<td style="width:175px;">BD Biosciences</td>
			<td style="width:119px;">611292</td>
			<td style="width:347px;">Mouse monoclonal, use at 1:20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">S96857</td>
			<td style="width:347px;">Lab grade, 95% denatured</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Forceps</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td>11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass coverslips</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">16004-330</td>
			<td>#1&#160; thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass microscope slides</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">12-550-15</td>
			<td>Standard glass slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Methanol</td>
			<td style="width:175px;">Fisher</td>
			<td style="width:119px;">A411</td>
			<td>Store in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Microcentrifuge tubes</td>
			<td style="width:175px;">VWR</td>
			<td style="width:119px;">20170-038</td>
			<td>capped tubes, not sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Normal goat serum</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">16210-064</td>
			<td>Aliquot 1ml tubes, store in freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Paraformaldehyde</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">P-6148</td>
			<td>Reagent grade, crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Phosphate buffered saline (PBS)</td>
			<td style="width:175px;">Quality Biological</td>
			<td style="width:119px;">119-069-131</td>
			<td>10X stock solution or made in lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Triton X-100</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:119px;">BP-151</td>
			<td>10% solution in water, store at room temp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tween-20</td>
			<td style="width:175px;">Life Technologies</td>
			<td style="width:119px;">85113</td>
			<td>10% solution in water, store at room temp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Compound microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:119px;">E-600</td>
			<td>Mount on vibration-free table</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">C1 Plus two-laser scanning confocal</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:119px;">C1 Plus</td>
			<td>Run by EZ-C1 program, but upgrades use &#34;Elements&#34;</td>
		</tr>
	</tbody>
</table>

immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos

ההתפשטות מהירה של תאים, ביטוי ספציפי הרקמות של גנים ואת הופעתה של רשתות איתות לאפיין התפתחות עוברית מוקדמת של כל בעלי החוליות. קינטיקה ומיקום של אותות - אפילו בתוך תאים בודדים - בעובר המתפתח משלים זיהוי של גנים התפתחותיים חשובים. טכניקות Immunostaining מתוארים כי הוכחו להגדיר את קינטיקה של אותות בעלי חיים תאיים ושלמים במבנים קטנים כמו cilia העיקרי. טכניקות לתיקון, הדמיה ועיבוד תמונות באמצעות מיקרוסקופ מתחם confocal-סריקת לייזר ניתן להשלים כמה כמו 36 שעות. דג הזברה (Danio rerio) הוא אורגניזם רצוי חוקרים המבקשים לבצע מחקרים בתוך בעלי החוליות היא הזולה רלוונטי מחלות אנושיות. knockouts או knockdowns הגנטיים חייב להיות מאושר על ידי האובדן של מוצר החלבון בפועל. אישור כזה של אובדן חלבוןיכול להיות מושג באמצעות הטכניקות המתוארות כאן. רמזים לתוך מסלולי איתות ניתן לפענח גם באמצעות נוגדנים כי הם תגובתי עם חלבונים ששונו שלאחר translationally ידי זרחון. שמירה ואופטימיזציה של המדינה פוספורילציה של אפיטופ לכן זה קריטי כדי קביעה זו לבין מושגת על ידי פרוטוקול זה. מחקר זה מתאר שיטות לתקן עוברים במהלך 72 שעות של פיתוח הראשון שיתוף בתרגום במגוון אפיטופים רלוונטי עם cilia בתוך שלפוחיות (KV) של Kupffer, הכליה ואת האוזן הפנימית. טכניקות אלו הם ישירים, אינם דורשים דיסקציה יכול להסתיים בתוך פרק זמן קצר יחסית של זמן. הקרנת ערימות תמונת confocal לתוך תמונה אחת היא אמצעי שימושי של הצגת נתונים אלה.

תנאים אופטימליים חזותי phospho-אפיטופים שיטות המתארות את immunolocalization של אפיטופים חלבון עוברי דג זברה כבר דלילות יחסית בהשוואה לאלו ללוקליזציה mRNAs באמצעות כלאה באתרה. Fixatives המשמשים לא?...

Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

טכניקות מתוארים כדי immunostain-אפיטופים phospho כולן עוברי דג הזברה ולאחר מכן לנהל לוקליזציה confocal ניאון בשני צבעים במבנים הסלולר קטן כמו cilia העיקרי. טכניקות לתיקון והדמיה יכול להגדיר את המיקום ואת קינטיקה של המראה או הפעלה של חלבונים ספציפיים.

Phospho-אפיטופים Immunostaining באיברים ריסי של עוברי דג הזברה שלמים הר

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Research

JoVE Journal

Chemistry

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Metabolomics לא ממוקדים ממקורות ביולוגיים באמצעות Ultraperformance כרומטוגרפיה נוזלית-רזולוציה גבוהה ספקטרומטריית מסה (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

מדידות דיוק אבץ איזוטופי גבוהות יישומים לאיברי עכבר

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Radiolabeling וכימות של רמות סלולריות של Phosphoinositides ידי נצנץ זרימה מצמיד כרומטוגרפיה נוזלית ביצועים גבוהים

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

רצף של הצמח קיר Heteroxylans שימוש enzymic, כימית (מתילציה) הפיזיקליים (ספקטרומטריית מסה, תהודה מגנטית גרעינית) טכניקות

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

סינתזה של חלבון Bioconjugates<em&gt; דרך</em&gt; כימיה ציסטאין-maleimide

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Accessing Valuable Ligand Supports for Transition Metals: A Modified, Intermediate Scale Preparation of 1,2,3,4,5-Pentamethylcyclopentadiene

גישה וליובל ליגנד עומדי מתכות מעבר: הכנת סולם שונה, ביניים של 1,2,3,4,5-Pentamethylcyclopentadiene

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

Microprobe נימי אלקטרופורזה ספקטרומטריית עבור מטבולומיקס תא בודד ב- Live צפרדע (צפרדע רפואית זריזה) העוברים

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of FÃƒÆ’Ã‚Â¢Ãƒâ€¹Ã¢â‚¬Â '

Photoelectron הדמיה של אניונים מאויר על ידי ניתוק 310 ננומטר F−

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

שינוי בהשראה ביו השטח Polydopamine של Nanodiamonds והצמצום שלה של סילבר חלקיקים

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-מבוסס על פעילות מבוססת על קביעת עיכוב מורכבים, IC50 ערכים, פעילות פרועובדתית, ואת הפעילות התא כולו של נוקלאוטילסייד הריבוליטיות

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...