Name: visualization and quantitative analysis of embryonic angiogenesis in xenopus tropicalis
Uploaded: 2017-05-25T14:00:00.000+00:00
Duration: 6 min 5 s
Description: Watch this Scientific Journal Video about Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis at JoVE.com

מחקר זה קיבל השראה מעבודתו של לוין ואח &#39;. , אשר תיאר שיטה זו ניסיוני וסיפק תיאור מקיף של פיתוח כלי הדם ב Xenopus laevis. אנו מודים לחברי המעבדה שלנו על הקלט שלהם. מחקר זה נתמך על ידי יוזמת המחקר המובילה של Yonsei University for Future of 2015 (2015-22- 0095) ותוכנית פיתוח הטכנולוגיה הביו-רפואית של הקרן הלאומית למחקר (NRF) הממומנת על ידי משרד המדע, ICT &amp; תכנון עתידי ( NRF-2013M3A9D5072551)

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למחברים אין מה לחשוף.

הפרוטוקול שהוצג כאן פותח לראשונה על ידי עלי H. Brivanlou ועמיתיו לחקור אירועים התפתחותיים במהלך היווצרות כלי הדם ב Xenopus laevis 4 , אבל, כפי שמוצג בכתב יד זה, זה יכול להיות מיושם על בעלי חיים קטנים אחרים. הזרקת דיו לתוך הלב הוא פשוט לבצע, ואת הרשת כלי הדם כולו יכו...

Vasculogenesis, היווצרות של כלי דם חדשים תאים אנדותל שנולדו לאחרונה, אנגיוגנזה, היווצרות של כלים חדשים מן הכלים הקיימים מראש, הם שני תהליכים שונים המעצבים את כלי הדם עובריים 1 . כל dysregulation בתהליכים אלה גורמת למחלות לב שונות וחריגות מבניות של כלי שיט. יתר על כן, הגידול קשורה לצמיחה כלי בלתי מבוקרת. ככזה, מנגנונים מולקולריים שבבסיס וסקולוגנזה ואנגיוגנזה הם נושא לחקירה אינטנסיבית 2 . Xenopus דג הזברה הם מודלים חוליות אטרקטיבי עבור vasculogenesis ומחקרים אנגיוגנזה, מכמה סיבות. ראשית, העוברים שלהם קטנים; לכן, קל יחסית לדמיין את כלי הדם כולו. שנית, התפתחות עובריים היא מהירה; זה לוקח רק כמה ימים על כל כלי הדם לפתח, שבמהלכן vascul המתפתח<html> בבית ניתן לדמות. שלישית, התערבויות גנטיות פרמקולוגיות לפני ובמהלך היווצרות כלי הם קלים לביצוע, כגון באמצעות microinjection של antisense morpholino נוקליאוטידים (MO) לתוך העובר המתפתח או באמצעות יישום אמבטיה של תרופות 3 , 4 , 5 . היתרון הייחודי של Xenopus על דג הזברה היא כי מניפולציות אמבריולוגיות ניתן לבצע כי Xenopus עוקב clevages holoblastic סטריאוטיפיים ואת מפת הגורל עובריים מוגדר היטב 6 . לדוגמה, ניתן ליצור עובר שבו צד אחד לרוחב רק מניפולציה גנטית על ידי הזרקת מו antisense לתא אחד בשלב שני תא. ניתן גם להשתיל את הלימבורד הלב מעובר אחד לאחר כדי לקבוע אם הגן מפעיל את תפקודו על ידי מנגנון פנימי או מהותי-תאהתחת = &quot;xref&quot;&gt; 7. למרות טכניקות אלו פותחו בעיקר Xenopus laevis , שהוא allotetraploid ולכן אינו אידיאלי עבור מחקרים גנטיים, הם יכולים להיות מיושמים ישירות Xenopus tropicalis , מינים דיפלואידי קרובים 8 . אחת הדרכים לדמיין את כלי הדם בעובר Xenopus לחיות היא להזריק צבע פלואורסצנטי לתווית את כלי הדם. Acetylated צפיפות נמוכה lipoprotein (AcLDL) שכותרתו עם מולקולת פלורסנט כגון DiI הוא בדיקה מאוד שימושי. שלא כמו lDL unacetylated, AcldL אינו קשור לקולטן LDL 9 אבל הוא endocytosed על ידי מקרופאגים ותאי אנדותל. הזרקת DiI-ACLDL אל הלב של חיה תוצאות חיה תיוג פלורסנט ספציפיים של תאים אנדותל, ואת כלי הדם כולו יכול להיות צילמו על ידי מיקרוסקופ פלואורסצנטי בעוברים חיים או קבוע 4 . הנה, אנחנו presEnt מפורט פרוטוקולים להדמיה וכימות של כלי הדם באמצעות DiI-AcLDL ב xenopus tropicalis ( איור 1 ). אנו מספקים נקודות מעשיות מרכזיות, עם דוגמאות של ניסויים מוצלחים ולא מוצלחים. בנוסף, אנו מספקים שיטה פשוטה לניתוח כמותי של המורכבות של כלי הדם, אשר עשוי להיות שימושי בהערכת ההשפעות של גורמים גנטיים וסביבתיים על עיצוב של רשת כלי הדם.

<table><tbody><tr><td>35 mm Petri dish</td><td>SPL</td><td>10035</td><td>Sylgard mold frame</td></tr><tr><td>60 mm Petri dish</td><td>SPL</td><td>10060</td><td>Embryo raising tray</td></tr><tr><td>Borosilicate Glass</td><td>Sutter instrument</td><td>B100-50-10</td><td>Needle for injection</td></tr><tr><td>BSA</td><td>Sigma</td><td>A3059-10G</td><td>Coating reagent</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>D.S.P.GR Reagent</td><td></td><td>0.1x MBS component</td></tr><tr><td>Coverslip</td><td>Superior</td><td>HSU-0111520</td><td>For confocal imaging</td></tr><tr><td>DiI-AcLDL</td><td>Thermo Fisher Scientific</td><td>L3484</td><td>Vessel staining solution</td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH.30919.02</td><td>For storage of testis</td></tr><tr><td>Fiber Optical Illuminator</td><td>World Precision Instruments</td><td>Z-LITE-Z</td><td>Light</td></tr><tr><td>Ficoll</td><td>Sigma</td><td>F4375</td><td>Injection buffer</td></tr><tr><td>Flaming/Brown Micropipette Puller</td><td>Sutter instrument</td><td>P-97</td><td>Injection needle puller</td></tr><tr><td>Forcep</td><td>Fine Science Tool</td><td>11255-20</td><td>For embryo hatching and&lt;br/&gt; needle tip cutting</td></tr><tr><td>Glass Bottom dish</td><td>SPL</td><td>100350</td><td>For confocal imaging</td></tr><tr><td>hCG</td><td>MNS Korea</td><td></td><td>For priming of frogs</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>Buffering agent</td></tr><tr><td>Incubator</td><td>Lab. Companion</td><td>ILP-02</td><td>For raising embryos</td></tr><tr><td>KCl</td><td>DAEJUNG</td><td>6566-4400</td><td>MBS component</td></tr><tr><td>L15 medium</td><td>Gibco</td><td>11415-114</td><td>For storage of testis</td></tr><tr><td>L-cysteine</td><td>Sigma</td><td>168149-100G</td><td>De-jellying reagent</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M7506</td><td>MBS component</td></tr><tr><td>Microtube</td><td>Axygen</td><td>MCT-175-C-S</td><td>For storage of testis</td></tr><tr><td>MS222</td><td>Sigma</td><td>E10521</td><td>Anesthetic powder</td></tr><tr><td>NaCl</td><td>DAEJUNG</td><td>7647-14-5</td><td>MBS component</td></tr><tr><td>NaOH</td><td>Sigma</td><td>S-0899</td><td>pH adjusting reagent</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td>Fixatives</td></tr><tr><td>PBS</td><td>BIOSESANG</td><td>P2007</td><td>Buffer for imaging</td></tr><tr><td>pH paper</td><td>Sigma</td><td>P4536-100EA</td><td>For confirming pH</td></tr><tr><td>PICO-LITER INJECTOR</td><td>Waner instruments</td><td>PLI-100A</td><td>For injection</td></tr><tr><td>Pin</td><td>Pinservice</td><td>26002-10</td><td>For incision</td></tr><tr><td>Pinholder</td><td>Scitech Korea</td><td>26016-12</td><td>For incision</td></tr><tr><td>Precision Stereo Zoom Binocular Microscope</td><td>World Precision Instruments</td><td>PZMIII</td><td>For visual screening</td></tr><tr><td>Standard Manual Control Micromanipulator&amp;nbsp;</td><td>Waner instruments</td><td>W4 64-0056</td><td>For microinjection</td></tr><tr><td>SYLGARD 184 Kit</td><td>Dow Corning</td><td></td><td>For DiI injection</td></tr><tr><td>Transfer pipette</td><td>Korea Ace Scientific Co.</td><td>YM.B78-400</td><td>For eggs and&lt;br/&gt; embryo collection</td></tr></tbody></table>

visualization and quantitative analysis of embryonic angiogenesis in xenopus tropicalis

כלי הדם מספקים חמצן וחומרים מזינים בכל הגוף, ויצירת רשת כלי הדם נמצאת תחת שליטה התפתחותית הדוקה. הדמיה יעילה של vivo של כלי הדם ואת כימות אמין של המורכבות שלהם הם המפתח להבנת ביולוגיה ומחלות של הרשת כלי הדם. כאן, אנו מספקים שיטה מפורטת לדמיין כלי דם עם צבע פלורסנט זמין מסחרית, פלזמה אנושית acetylated צפיפות נמוכה lipoprotein DiI מורכבים (DiI-AcLDL), וכמות המורכבות שלהם tropis Xenopus . כלי דם יכול להיות מתויג על ידי הזרקת פשוטה של ​​DiI-AcLDL לתוך הלב פועם של העובר, וכלי הדם העובר כולו יכול להיות צילמו עוברי לחיות או קבוע. בשילוב עם הפרעות גנטיות על ידי המיקרו-זרקור הממוקד של חומצות גרעין ו / או יישום אמבט של ריאגנטים פרמקולוגיים, תפקידים של גן או של מסלול איתות על התפתחות כלי הדם יכולים להיות inveStigated בתוך שבוע מבלי להזדקק חיות מתוחכמות מהונדסים גנטית. בגלל מערכת ורידים מוגדרת היטב של קסנופוס ואת אנגיוגנזה סטריאוטיפית שלה, הנבטה של ​​כלי קיים מראש, מורכבות כלי ניתן לכמת ביעילות לאחר ניסויים ההפרעה. זה פרוטוקול פשוט יחסית צריך לשמש כלי נגיש בקלות בתחומים מגוונים של מחקר לב וכלי דם.

ציר הזמן של ניסויים (תרשימים 1 ו -2) זמן קצר לאחר ההפריה, ממוקד microinjection יכול להתבצע כדי לווסת ביטוי גנים. לדוגמה, antisense MO אשר נקשר ספציפית קודון חניכה של mRNA Tie2 אנדוגני יכול להיות מוזרק, מעכב את התרגום...

Watch this Scientific Journal Video about Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis at JoVE.com

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

פרוטוקול זה מדגים שיטה מבוססת הקרינה לדמיין את כלי הדם וכדי לכמת את המורכבות שלה tropis Xenopus . כלי הדם ניתן הדמיה דקות לאחר הזרקת צבע פלואורסצנטי לתוך הלב הפועם של עובר לאחר מניפולציות גנטיות ו / או תרופתי ללמוד התפתחות הלב וכלי הדם in vivo .

ויזואליזציה וניתוח כמותי של אנגיוגנזה עובריים ב<em&gt; Xenopus טרופי</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

visualization-quantitative-analysis-embryonic-angiogenesis-xenopus

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Developmental Biology

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

8.4K Views.  Yonsei University College of Medicine.  פרוטוקול זה מדגים שיטה מבוססת הקרינה לדמיין את כלי הדם וכדי לכמת את המורכבות שלה tropis Xenopus . כלי הדם ניתן הדמיה דקות לאחר הזרקת צבע פלואורסצנטי לתוך הלב הפועם של עובר לאחר מניפולציות גנטיות ו / או תרופתי ללמוד התפתחות הלב וכלי הדם in vivo

Video: Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

הבנת organogenesis המוקדם שימוש פשוטה<em&gt; באתר</em&gt; הכלאת פרוטוקול ב<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

באמצעות ניתוח של Confocal<em&gt; Laevis Xenopus</em&gt; לחקירת מאפננים של עירובים מורפוגן Wnt וששש

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

כמו תא גזע<em&gt; Xenopus</em&gt; Explants העובריים לחקר תכונות המוקדמות עצבי התפתחותית<em&gt; במבחנה</em&gt; ו<em&gt; In vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

טכניקה לכוון Microinjection אל פיתוח<em&gt; Xenopus</em&gt; כליה

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

בVivo הדמיה וככמת של התגובה המארחת אנגיוגנטית ב-Zebrafish גידול Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Cancer Research

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

הר שלם בתוך היברידיזציה ב-Zebrafish עוברים ושיטת היווצרות צינור ב-iPSC-ECs ללמוד את התפקיד של אנגלין בפיתוח כלי דם

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-תא הדמיה וניתוח כמותי של תאים אפיתל עובריים ב Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Biology

שימוש בדגי זברה זחלים למחקרים יעילים בגידולי Vivo

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

מחבר זרקור: קידום טיפולים בסרטן באמצעות השתלת קסנו-גידול בדגי זברה

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

שיטה פשוטה, מהירה ויעילה לניתוח השתלת גידול בעוברים שקופים של דגי זברה

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

ויזואליזציה וניתוח כמותי של אנגיוגנזה עובריים ב

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Chapters in this video

הבנת organogenesis המוקדם שימוש פשוטה

באמצעות ניתוח של Confocal

כמו תא גזע

טכניקה לכוון Microinjection אל פיתוח

בVivo הדמיה וככמת של התגובה המארחת אנגיוגנטית ב-Zebrafish גידול Xenografts

הר שלם בתוך היברידיזציה ב-Zebrafish עוברים ושיטת היווצרות צינור ב-iPSC-ECs ללמוד את התפקיד של אנגלין בפיתוח כלי דם

Live-תא הדמיה וניתוח כמותי של תאים אפיתל עובריים ב Xenopus laevis

שיטה פשוטה, מהירה ויעילה לניתוח השתלת גידול בעוברים שקופים של דגי זברה

שיטה פשוטה, מהירה ויעילה לניתוח השתלת גידול בעוברים שקופים של דגי זברה

שיטה פשוטה, מהירה ויעילה לניתוח השתלת גידול בעוברים שקופים של דגי זברה