This project was supported by a Leon J. Thal SEED grant from the California Institute for Regenerative Medicine. Tobias Henning was funded by a Research stipend from the German Research Association (DFG, HE 4578/1-2). We want to gratefully acknowledge Juanito Meneses for his advice on the culture of human embryonic stem cells.

Labeling of mesenchymal stem cells with the fluorescent dye DiD
<ol>
<li>To begin the procedure for labeling mesenchymal stem cells, trypsinize and count the cells to get a suspension with a defined number of cells. </li>
<li>Take the cells out of the incubator and aspirate out old media from the flasks containing the cells to be labeled </li>
<li>Wash the cells with 10 ml of Mg/Ca-free PBS. Aspirate out the PBS. The Mg and Ca would inhibit the Trypsin, therefore we use PBS without these.</li>
<li>Add pre-warmed 0.05% Trypsin, for a T75 flask we use 5ml. Ensure that the entire surface of the flask is covered.</li>
<li>Incubate at 37&deg;C in the incubator for about 5 minutes.</li>
<li>Confirm the detachment under the microscope. If the cells still adhere to the flask, tap it a few times and wait a little longer until the trypsinization is successful.</li>
<li>Now, it is necessary to neutralize the Trypsin by adding media containing 10% FCS. We use an equal amount of media as there is Trypsin. </li>
<li>Pipette up and down a few times to ensure that all the cells are re-suspended in the media.</li>
<li>Transfer the cell solution to a sterile 15ml capped polypropylene tube.</li>
<li>Centrifuge at 400 rcf for 5 minutes.</li>
<li>Aspirate out the supernatant ensuring not to disturb the cell pellet.</li>
<li>Resuspend the cells in DMEM and proceed with the cell count.</li>
<li>Suspend the cells to be labeled at a density of 1x10^6 per ml in serum-free culture medium (DMEM). </li>
<li>This was all preparation of the cell suspension. Now, it is ready to start the labeling.</li>
<li>First, add 5 &micro;L of DiD contrast agent per ml of cell suspension.</li>
<li>Then, mix the solution by gently pipetting.</li>
<li>Incubate the cells with the labeling solution at 37&deg;C for 20 minutes in a 6-well low-attachment dish. </li>
<li>Once the simple incubation is complete, it is necessary to transfer the cell solution to a 15 ml capped polypropylene tube.</li>
<li>Centrifuge it down at 400 rcf for 5 minutes.</li>
<li>Aspirate out the labeling medium, ensuring not to disturb cell pellet. </li>
<li>Wash the cells with PBS. Pipette the cells up and down, making sure to break up the cell pellet.</li>
<li>Repeat the latter two steps two more times, so there is a total of 3 washing steps.</li>
<li>Count the cells and perform a Trypan blue test to determine cell viability. These are standard cell culture protocols that we are not going to explain here.</li>
<li>Your cells are now ready to be imaged in optical imager.</li>
</ol>
Labeling of human embryonic stem cells with the fluorescent dye ICG
<ol>
<li>To begin the procedure for labeling human embryonic stem cells, prepare the contrast agent Indocyanine Green. Mix it with the transfection agent Protamine.</li>
<li>Measure out 1mg of the Indocyanine Green powder. Dissolve the ICG powder in 100 &micro;L of Dimethyl Sulfoxide (DMSO).</li>
<li>Add 400 &micro;L of Dulbecco's Modified Eagles Medium (DMEM + 10% Fetal Calf Serum) media to the mixture and shake it well. This results in a final concentration of 2mg/ml of Indocyanine Green.</li>
<li>Add the transfection agent Protamine. Protamine acts as a shuttle for the contrast agent, so that it gets into the cell more efficiently.</li>
<li>Mix 5 &micro;L Protamine Sulfate, which is supplied at a concentration of 10mg/ml, with 300 &micro;L ICG and 300 &micro;L serum free Dulbecco's Modified Eagles Medium. </li>
<li>Gently shake the new transfection solution for 5 minutes to allow complex to form.</li>
<li>The labeling solution is ready.</li>
<li>Aspirate out the old media from hESC 10mm Petri dish.</li>
<li>Add 5ml of pre-warmed serum-free DMEM.</li>
<li>Add the previously prepared Protamine/ICG solution to the cells. Start the 1 hour incubation by placing the dish in an incubator at 37&deg;C.</li>
<li>After the incubation is complete, remove the dish from the incubator and aspirate out the labeling solution.</li>
<li>Wash the cells by rinsing the dish with 5 ml PBS. </li>
<li>Aspirate out the PBS and replace with 5ml of 0.25% Trypsin. Incubate the dish at 37&deg;C for 5 minutes to allow trypsinization to occur. It helps to shake the dish a little every once in a while.</li>
<li>Gently pipette up and down, to break up the remaining colonies.</li>
<li>Neutralize the Trypsin by adding an equal amount of KSR to the dish.</li>
<li>Transfer the cell solution to a 15ml tube and centrifuge the solution at 400 rcf for 5 minutes.</li>
<li>Resuspend the cells in full media.</li>
<li>If there still are clumps at this point,&nbsp;trypsinize and centrifuge again.</li>
<li>Once a&nbsp;clump-free cell solution is achieved, aspirate out the old media and resuspend the pellet in 10ml of pre-warmed full ESC media.</li>
<li>At this point, it is necessary to separate the mouse feeder cells from the hESCs. This is done to ensure that, later, only imaging the stem cells occurs.</li>
<li>For this, transfer the cell solution to a gelatin coated 10 cm dish. </li>
<li>Put the dish into the 37&deg;C incubator and let it sit for 45 minutes.&nbsp;During this time, make sure not to disturb the dish.&nbsp; Now, the feeders will adhere to the dish and the stem cells will not.</li>
<li>Transfer the solution out of the Petri dish. and now we have a labeled single cells solution of human embryonic stem cells. </li>
<li>The cells can now be counted and a Trypan blue test can be performed on them.</li>
<li>The&nbsp;cells are ready to be imaged!</li>
</ol>
&nbsp;

<ol>
	<li>Daldrup-Link, H. E., Rudelius, M., Metz, S., Piontek, G., Settles, M., Pichler, B., Heinzmann, U., Weinmann, H. J., Schlegel, J., Link, T. M., Rummeny, E. J., Oostendorp, R. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+tracking+with+Gadophrin-2+--+a+bifunctional+contrast+agent+for+MR+imaging,+optical+imaging+and+fluorescence+microscopy.">Stem cell tracking with Gadophrin-2 -- a bifunctional contrast agent for MR imaging, optical imaging and fluorescence microscopy.</a> Eur J Nucl Med Mol Imaging. 31, 1312-1321 (2004).</li><li>Simon, G. H., Daldrup-Link, H. E., Kau, J., Metz, S., Schlegel, J., Piontek, G., Saborowski, O., Demos, S., Duyster, J., Pichler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+experimental+arthritis+using+allogeneic+leukocytes+labeled+with+a+near-infrared+fluorescent+probe.">Optical imaging of experimental arthritis using allogeneic leukocytes labeled with a near-infrared fluorescent probe.</a> Eur J Nucl Med &amp; Mol Imaging. 33, 998-1006 (2006).</li><li>Sutton, E., Henning, T., Pichler, B., Bremer, C., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Tracking+with+Optical+imaging.">Cell Tracking with Optical imaging.</a> Eur Radiol. 2, 350-357 (2003).</li><li>Funovics, M. A., Alencar, H., Su, H. S., Khazaie, K., Weissleder, R., Mahmood, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+multichannel+near+infrared+endoscope+for+mouse+imaging.">Miniaturized multichannel near infrared endoscope for mouse imaging.</a> Mol Imaging. 2, 350-357 (2003).</li></ol>

OI is a relatively new imaging technique, based on the detection of fluorescence. OI is as sensitive as radiotracer-based imaging techniques, but not associated with any irradiation exposure. OI provides an effective means of tracking cells non-invasively and repetitively, thereby providing insight into cell migration to the target site. One major limitation of the technique is the limited tissue penetration of fluorescent probes in vivo. Thus, a tracer accumulation in deep tissues, more than about 5-10 cm from the skin ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Indocyanine Green (IR-125)</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>AC41254-1000 </td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D-2650 </td>
<td>&nbsp;</td>
<tr>
<td>D-MEM High Glucose </td>
<td>Reagent</td>
<td>Sigma</td>
<td>D5648</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G1890</td>
<td>Dilute to final concentration of 0.1% in PBS</td>
</tr>
<tr>
<td>PBS, Mg and Ca free</td>
<td>Reagent</td>
<td>GIBCO </td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA 0.05% </td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25300-120</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA 0.25% </td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25200-114	</td>
<td>&nbsp;</td>
<tr>
<td>FCS, characterized</td>
<td>Reagent</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Cell Strainer (40 &#x03BC;m Nylon)</td>
<td>Reagent</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>DiD</td>
<td>Reagent</td>
<td>Molecular Probes</td>
<td>V-22887</td>
<td>
</td>
</tr>
<tr>
<td>Knockout DMEM</td>
<td>Reagent</td>
<td>GIBCO</td>
<td>10829018</td>
<td>&nbsp;</td>
<tr>
<td>Knockout Serum Replacer</td>
<td>Reagent</td>
<td>GIBCO</td>
<td>10828028</td>
<td>
</td>
</tr>
<tr>
<td>b-Mercaptoethanol </td>
<td>Reagent</td>
<td>Sigma</td>
<td>7522</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential Amino Acids </td>
<td>Reagent</td>
<td>GIBCO</td>
<td>11140050</td>
<td>&nbsp;</td>
<tr>
<td>FGF-2</td>
<td>Reagent</td>
<td>R&D Systems </td>
<td> 233-FB-025</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine 200mM </td>
<td>Reagent</td>
<td>GIBCO</td>
<td>25030081</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>Reagent</td>
<td>GIBCO</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Protamine Sulfate</td>
<td>Reagent</td>
<td>American Pharmaceutical Partners Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

labeling stem cells with fluorescent dyes for non-invasive detection with optical imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

Watch this Scientific Journal Video about Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging at JoVE.com

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

labeling-stem-cells-with-fluorescent-dyes-for-non-invasive-detection

Research

JoVE Journal

Biology

13.3K Aufrufe.  Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco. Dieses Video zeigt Techniken für die Kennzeichnung von menschlichen embryonalen Stammzellen und mesenchymalen Stammzellen mit fluoreszierenden Farbstoffen. Diese Technik kann für eine in vivo Verfolgung der transplantierten Stammzellen mit optischen Bildgebung und zur histopathologischen Korrelation mit Fluoreszenzmikroskopie eingesetzt werden.

Serie: Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.