Name: multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos
Uploaded: 2017-08-09T15:00:00
Description: Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

Die Autoren danken Thomas Schilling für freundlicherweise gifting Tg (Sox10:eGFP) Fisch und Mary Halloran für freundlicherweise gifting Tg(Foxd3:GFP) Fisch.

The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA).
1. Embryo Collection for Time-lapse Imaging
<ol>
	<li>Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating. 
	NOTE: The Tg(sox10:EGFP) and Tg(foxd3:G...

<ol>
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Multi-Photonen-Zeitraffer-Bildgebung ermöglicht die in Vivo Verfolgung von Transienten und wandernden Zellpopulationen. Diese leistungsstarke Technik kann verwendet werden, um embryonale Prozesse in Echtzeit zu studieren, und in der vorliegenden Studie, die Ergebnisse dieser Methode erweitert das aktuelle Wissen der Neuralleiste Zelle Migration und Entwicklung. Time-Lapse bildgebende Studien nutzen in der Regel konfokale Laser-scanning-Mikroskopie. 29 , 30</s...

Angeborene Augenkrankheiten können dazu führen, dass Blindheit im Kindesalter und sind häufig durch Anomalien der kranialen Neuralleiste. Zellen der Neuralleiste sind vorübergehende Stammzellen, die aus dem Neuralrohr entstehen und bilden zahlreiche Gewebe im gesamten Körper. 1 , 2 , 3 , 4 , 5 Zellen der Neuralleiste, Prosencephalon und Mittelhirnes, abgeleitet ergeben sich die Knochen und Knorpel des Mittelgesichts und frontalen Regionen, und die Iris, Hornhaut, trabekuläre Geflecht und Sklera im vorderen Bereich des Auges. 4 , 6 , 7 , 8 Neuralleiste Zellen aus Rhombencephalon Form, die den Rachen Bögen, Kiefer und kardiale Ausflusstrakt. 1 , 3 , 4 , 9 , 10 Studien haben die Beiträge von der Neuralleiste, okuläre und Periorbitalregion Entwicklung hervorgehoben, betonend, wie wichtig dieser Zellen im vertebrate Augenentwicklung. In der Tat, Störung der Neuralleiste Zellwanderung und Differenzierung führen zu okuläre und kraniofaziale Anomalien wie in Axenfeld-Rieger-Syndrom und Peters-Plus-Syndrom festgestellt. 11 , 12 , 13 , 14 , 15 , 16 , 17 so, wird ein umfassendes Verständnis von Migration, Proliferation und Differenzierung dieser Zellen der Neuralleiste Einblick in die Komplexität der zugrunde liegenden angeborene Augenkrankheiten, zu schaffen.
Der Zebrabärbling ist ein leistungsfähiges Modellorganismus für okuläre Entwicklung zu studieren, wie die Strukturen des Auges Zebrafisch Pendants Säugetier-ähneln und viele Gene evolutionär zwischen Zebrafisch und Säugetiere konserviert sind. 18 , 19 , Darüber hinaus sind 20 Zebrafisch-Embryonen transparent und ovipar, erleichtert die Visualisierung der Augenentwicklung in Echtzeit.
Erweiterung auf zuvor veröffentlichte Arbeit,6,7,20 das wandernde Muster der Zellen der Neuralleiste wurde beschrieben mit Multi-Photonen Fluoreszenz Zeitraffer imaging in transgenen Zebrafisch Zeilen beschriftet mit grün fluoreszierendes Protein (GFP) von SRY (Geschlecht-Bestimmung der Region Y) transcriptional kontrolliert-Box 10 (sox10) oder Forkhead-Box-D3 (foxd3) gen regulatorischen Regionen. 21 , 22 , 23 , 24. Zeitraffer Multi-Photonen-Fluoreszenz-Bildgebung ist eine leistungsstarke Technik, die die fortschrittliche optische Techniken der Laser-scanning-Mikroskopie mit langen Wellenlänge Multi-Photonen Fluoreszenzanregung hochauflösende, dreidimensionale Aufnahmen der Proben mit dem Stichwort Fluorophore kombiniert. 25 , 26 , 27 die Verwendung von Multi-Photonen-Laser hat deutliche Vorteile gegenüber standard konfokalen Mikroskopie, einschließlich erhöhte Gewebe eindringen und verminderte Fluorophore bleichen.
Mit dieser Methode wurden zwei verschiedene Populationen von Zellen der Neuralleiste unterschiedlicher Zeitpunkt der Migration und wandernde Wege Neuralleiste diskriminierte, nämlich foxd3-positiven Zellen in der Periorbitalregion Mesenchym und entwickelnde Auge und Neuralleiste sox10-positiven Zellen in der kraniofazialen Mesenchym. Mit dieser Methode wird ein Konzept für die Migration von okulären und kraniofaziale Neuralleiste Migration im Zebrafisch visualisieren eingeführt, macht es einfach, regulierten Neuralleiste Migration in Echtzeit während der Entwicklung zu beobachten.
Dieses Protokoll enthält Informationen zur Erzeugung von Zeitraffer-Videos während der frühen Augenentwicklung in Tg (Sox10:EGFP) und transgenen Zebrafisch Tg (Foxd3:GFP), als Beispiel. Dieses Protokoll kann weiter für die hochauflösende, dreidimensionale, Echtzeit-Visualisierung der frühen Entwicklung der okulären und kraniofaziale Struktur abgeleitet aus Zellen der Neuralleiste im Zebrafisch angewendet werden. Darüber hinaus kann diese Methode für die Darstellung der Entwicklung der anderen Gewebe und Organe im Zebrafisch und anderen Tiermodellen weiter angewendet werden.

<table border="0" cellpadding="0" cellspacing="0" width="919">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Breeding Tanks with Dividers</td>
			<td style="width:175px;">Aquaneering</td>
			<td style="width:104px;">ZHCT100</td>
			<td style="width:391px;">Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">M205 FA Combi-Scope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;">Stereofluorescence Microscope - FusionOptics and TripleBeam</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">7760-5KG</td>
			<td style="width:391px;">Double PE sack. CAS No. 7647-14-5, EC Number 231-598-3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Potassium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">1049380500</td>
			<td style="width:391px;">Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Calcium Chloride Dihydrate</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">C79-500</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 10035-04-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Magnesium Sulfate (Anhydrous)</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">MX0075-1</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methylene Blue</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">284-12</td>
			<td style="width:391px;">Glass bottle; 25 g. Powder, Certified Biological Stain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Bicarbonate</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">SX0320-1</td>
			<td style="width:391px;">Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">N-Phenylthiourea</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">P7629-25G</td>
			<td style="width:391px;">&#62;98%. CAS Number 103-85-5, EC Number 203-151-2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Dimethylsulfoxide</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">D8418-500ML</td>
			<td style="width:391px;">Molecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tricaine Methanesulfonate</td>
			<td style="width:175px;">Western Chemical Inc.</td>
			<td style="width:104px;">MS222</td>
			<td style="width:391px;">Tricaine-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Low-Melt Agarose</td>
			<td style="width:175px;">ISC Bioexpress</td>
			<td style="width:104px;">E-3112-25</td>
			<td style="width:391px;">GeneMate Sieve GQA Low Melt Agarose, 25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Open Bath Chamber</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">RC-40HP</td>
			<td style="width:391px;">High Profile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glass Coverslips</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">12-545-102</td>
			<td style="width:391px;">Circle cover glass. 25 mm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">High Vacuum Grease</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">14-635-5C</td>
			<td style="width:391px;">2.0-lb. tube. DOW CORNING CORPORATION 
			1658832</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Quick Exchange Platform</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">QE-1</td>
			<td style="width:391px;">35 mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Stage Adapter</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">SA-20LZ-AL</td>
			<td style="width:391px;">16.5 x 10 cm</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:251px;">TC SP5 MP multi-photon microscope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mai Tai DeepSee Ti-Sapphire Laser</td>
			<td style="width:175px;">SpectraPhysics</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Laser Safety Box</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Leica Application Suite X (LAS X)&#160; Software</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;"></td>
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			<td height="42" style="height:42px;width:251px;">Photoshop CS 6 Version 13.0 x64 Software</td>
			<td style="width:175px;">Adobe</td>
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			<td style="width:391px;"></td>
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			<td height="21" style="height:21px;width:251px;">iMovie Version 10.1.4 Software</td>
			<td style="width:175px;">Apple</td>
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multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos

Angeborene Auge und kraniofaziale Anomalien reflektieren Störungen in der Neuralleiste, eine vorübergehende Bevölkerung von wandernden Stammzellen, die Anlass zu zahlreichen Zelltypen im ganzen Körper. Verständnis der Biologie der Neuralleiste beschränkt wurde, spiegelt einen Mangel an genetisch gefügig Modelle, die studierte in Vivo und in Echtzeit. Zebrafisch ist ein besonders wichtig Entwicklungs Modell für das Studium wandernde Zellenbevölkerungen, wie der Neuralleiste. Um Neuralleisten-Migration in der dritten Auge zu untersuchen, wurde eine Kombination der erweiterten optischen Techniken der Laser-scanning-Mikroskopie mit langwelligen Multi-Photonen Fluoreszenzanregung implementiert, um hochauflösende, dreidimensionale, Echtzeit-Videos des dritten Auges in den transgenen Zebrafish Embryos, nämlich Tg (Sox10:EGFP) und Tg (Foxd3:GFP), zu erfassen wie sox10 und foxd3 , in zahlreichen Tiermodellen gezeigt haben zu frühen Neuralleiste Differenzierung regulieren und wahrscheinlich Marker für Zellen der Neuralleiste darstellen. Multi-Photonen Zeitraffer Bildgebung wurde verwendet, um das Verhalten und die Migrationsmuster zwei Neuralleiste Zellpopulationen zur frühen Augenentwicklung zu erkennen. Dieses Protokoll enthält Informationen zur Erzeugung von Zeitraffer-Videos während der Zebrafisch Neuralleiste Migration als Beispiel und kann weiter angewendet werden, um die frühe Entwicklung von vielen Strukturen in dem Zebrafisch und sonstige Modellorganismen zu visualisieren.

Time-Lapse Multi-Photonen-Fluoreszenz-Bildgebung erzeugt eine Reihe von Videos, die die Migrationsmuster der kranialen Neuralleiste Zellen aufgedeckt, die zu der kraniofazialen Strukturen und vorderen Segments des Auges in der Tg (Sox10:EGFP) und Tg (Foxd3:GFP) Zebrafisch Linien führen. Als Beispiel Wandern sox10 -positive Neuralleiste Zellen zwischen 12 und 30 hpf vom Rand des Neuralrohrs in der kraniofazialen Region (Video 1, <strong class="...

Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Multi-Photonen-Time Lapse Imaging Entwicklung in Echtzeit zu visualisieren: Visualisierung der Migration Neuralleisten-Zellen in den Zebrafish Embryos

Eine Kombination der erweiterten optischen Techniken der Laser-scanning-Mikroskopie mit langen Wellenlänge Multi-Photonen Fluoreszenzanregung wurde implementiert, um hochauflösende, dreidimensionale, Echtzeit-Bildgebung der Neuralleiste Migration mit Tg (Sox10:EGFP) und Tg (Foxd3:GFP) Zebrafish Embryos zu erfassen.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

The overall goal of multi-photon time lapse imaging is to capture a high-resolution three dimensional real time images of migratory cell populations. This method can help answer key questions in developmental biology, such as mapping migration pathways of different cell populations. The main advantage of this technique over traditional confocal microscopy is that multi-photon lasers have deeper tissue penetration and decreased phototoxicity.This increases the usability in thicker tissues and allows for longer periods of image acquisition. To set up for embryo collection between 3:00 and 9:00 p.m. assemble breeding tanks and fill them with reverse osmosis, or RO water.Transfer up to three females and three males into opposing sides of the divided tank. The next morning, remove the divider shortly after the lights turn on in the vivarium. Allow undisturbed mating for 20 minutes or until sufficient numbers of embryos are produced.The embryos will be at the bottom of the breeding tank. When eggs are observed, lift the slotted inner tank out along with the fish, and quickly place them into a clean, solid outer tank filled with RO water. Use an egg collecting screen to collect eggs from the outer tank and transfer the eggs to Petri dishes containing 30 milliliters of 1X embryo medium.Incubate the collected eggs at 28.5 degrees Celsius. For transgenic embryos, at 24 hours post-fertilization or HPF, remove the dead eggs and assess the developmental stage of the embryos. Using forceps, remove the chorions from three to four embryos that express GFP.Under a dissecting fluorescence stereo microscope with a standard 460 to 490 nanometer band pass excitation filter, screen for GFP positive embryos. Transfer these embryos to a separate Petri dish containing 30 milliliters of fresh 1X embryo medium. After 20 HPF, place the screened and dechorionated GFP positive embryos in 0.003%PTU solution to inhibit pigmentation.Avoid initiating treatment earlier as it can have adverse effects on neural crest and neuroepithelial development. Add 0.4 grams of LowMelt Agarose powder to 20mL of 1x embryo medium. Heat the mixture for one to two minutes or until the solution is clear and all particles are dissolved.Aliquot approximately 1mL of the Agarose solution into fresh 1.5mL centrifuge tubes for storage. To set up the open bath chamber, place a small amount of high vacuum grease on the base of the open bath chamber. Place a circular glass cover slip onto the base and screw the top of the open bath chamber onto the base until it is tight.Pipette approximately 500-700 microliters of 2%Agarose solution into the open bath chamber until the base is approximately 3/4s filled. Wait 30 seconds to allow the Agarose to cool slightly without completely polymerizing and subsequently transfer a single embryo to the center of the base. Under a fluorescent stereo microscope, use a 10 microliter micro pipette tip to orient the embryo and position it near the bottom of the Agarose so that it doesn't float away when the embryo medium is added.A critical step is to make sure that the embryo is positioned correctly in the Agarose. In these experiments, we orient the embryo laterally but the embryo can also be positioned to focus on dorsal and ventral areas. Monitor and reposition the embryo until the Agarose has set.Then use a transfer pipette to entirely fill the assembled open chamber with time-lapse embryo medium. After placing the entire set up onto the stage of the microscope, according to the text protocol, use the five times objective to locate the embryo. Then manually raise the stage to the highest position and use the fine focus to position the embryo in the middle of the microscope range.Manually lower the stage and change the five time objective to the 25 time water immersion objective. Carefully raise the stage to bring the embryo back into focus. In the software, click on the XYTZ mode for obtaining multiple images at time or T intervals in the XY plane over a depth of Z.Use the epifluorescence or bright field view to find the depth of focus in the area of interest which will demarcate the z-stack.For these experiments, the lateral edge of the eye is the beginning of the z-stack. When focused here, in the software click the begin button. After focusing down to the mid-line of the embryo, which is the end of the z-stack, click the end button.A critical step is to make sure that the Z step encompasses the area of interest. In our case, we want to capture the width of the eye from the lateral to the medial edges. Click on the menu for adjusting the acquisition time and the imaging frequency.For adequate recovery of the fluor4 and survival of the embryo, allow for a ratio of at least one to three between z-stack acquisition when laser power is on and recovery time when laser power is off. With appropriate time for embryo recovery, set the time between z-stacks in the designated window. Then set the total length of time for the experiment in the appropriate window.Now, turn on the live image setting to make final adjustments to the laser settings. Adjust laser transmission, gain and offset slider bars within the software to optimize the fluorescence image. Also, adjust the orientation of the embryo as needed depending on the length of the experiment, anticipated growth of the embryo, et cetera.Make sure that the area of interest remains within the frame through the duration of the experiment. During time-lapse acquisition, turn off the epifluorescence light source, cover the stage with the laser safety box which is adequate for protection against background light, then press start. Access the open bath chamber through the sliding doors on the laser safety box to refill it with time lapse embryo medium every 8-12 hours.Following imaging acquisition, open the files in the image processing software, highlight the correct image series. In the software, choose the process menu, click on 3D deconvolution and apply to deconvolve each z-stack. Import individual tif files into video processing software, then select all tif files and drag them into the video editor.Adjust the length of each image within the video to 0.1 seconds. Finally, export the video as a mov or mp4 file. Seen here by multi-photon fluorescence imaging are the migration patterns of cranial neural crest cells that give rise to the cranial facial structures and anterior segment of the eye in transgenic sox10:EGFP and foxd3:GFP zebrafish lines.Sox 10 positive neural crest cells between 12 and 30 hpf migrate from the edge of the neural tube into the cranial facial region. The cells from the prosencephalon and mesencephalon migrate dorsally and ventrally to the developing eye to populate the periocular mesencine. As indicated here, the sox10 positive cells form the frontal nasal process.Neural crest cells from the rhombencephalon migrate ventrally and give rise to the pharyngeal arches. Time lapse imaging of foxd3 positive neural crest cells between 30 and 60 hpf show that these cells migrated between the optic cup and surface ectoderm and though the ocular fissure marked by the arrow and coalesced around the lens marked by the asterisk forming the iris stroma. Once mastered, this technique from start to finish can be done in four days if it is performed properly.When attempting this procedure, it is important to remember to choose embryos with a high amount of fluorescence. Setting up the software initially can be challenging but once the parameters are determined, additional changes are typically minimal. After watching this video, you should have a good understanding of how to use multi-photon time-lapse microscopy to image migrating cells in the embryo.

Research

JoVE Journal

Developmental Biology

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing<em&gt; In Vivo</em&gt; Zellmigration unter Verwendung von Zelltransplantationen und Zeitraffer-Imaging in Zebrafischembryonen

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Forschung

Entwicklungsbiologie

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Analyse von Zebrabärbling Nierenentwicklung mit Zeitraffer-Imaging einem Binokular Mit Ausgerüstet für Optische Schnitte

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Kinetische Messung und Echtzeit-Visualisierung von Somatic Reprogrammierung

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Induktion von Hypoxie im lebenden Frosch und Zebrafisch Embryonen

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Entwicklung eines In-vitro- Assay, hämatopoetischen Stammzellen und Vorläuferzellen Quantitate Zellen (HSPCs) bei der Entwicklung von Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Echtzeit Live-Zelle Durchflusszytometrie, Calcium Zustrom, Porenbildung und Phagozytose durch P2X7-Rezeptoren im Erwachsenen neurale Vorläuferzellen zu untersuchen

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Eine geschichtete Montagemethode für die verlängerte Zeitraffer-Konfokalmikroskopie ganzer Zebrafisch-Embryonen

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Visualisierung des sich entwickelnden Gehirns bei lebenden Zebrafischen mit Brainbow und Zeitraffer-Konfokalbildnis

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

Isolation, Kultur und adipogene Induktion von Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...