We thank David Theilmann (Pacific Agri-Food Research Centre, Summerland, British Columbia) for providing the baculovirus AcMNPV and for helpful discussion.
This work was supported by grants from the Canada Foundation for Innovation (CFI), the Canadian Institutes of Health Research (CIHR), the Michael Smith Foundation for Health Research (MSFHR), and the Natural Sciences and Engineering Research Council of Canada (NSERC).

Part 1: Preparation of Xenopus oocytes for microinjection
<ol>
	<li>Place a small piece (about 2 cm) of Xenopus laevis ovary into a 50-ml conical tube containing 20 ml of collagenase solution (5 mg/ml collagenase in calcium-free modified Barth&#8217;s saline: 88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 10 mM Hepes, pH 7.5). Collagenase is used to remove the follicle cells which surround the oocytes.</li>
	<li>Place the tube on a shaker platform and rock it gently (at 100 RPM) for one hour. This time varies with different lots of collagenase. Thus after one hour, take a small sample of oocytes and examine them under a dissecting microscope. Properly de-folliculated oocytes should be well separated from one another, and it should be easy to insert a glass needle into the oocyte. If the oocytes are not sufficiently de-folliculated, they are left in the collagenase solution and checked again every five minutes.</li>
	<li>Wash the oocytes three times with modified Barth&#8217;s saline (MBS: 88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 10 mM Hepes, pH 7.5), and transfer them to a 100-mm diameter Petri dish containing MBS plus 1% penicillin/streptomycin. The oocytes are now ready to be microinjected. If microinjection will be performed on another day, store the oocytes at 4 &#176;C for up to one week.</li>
	<li>Using a dissecting microscope, select mature stage VI oocytes for microinjection. These oocytes are large, with good contrast between the black animal hemisphere and the creamy-colored vegetal hemisphere.</li>
	<li>Transfer the mature stage VI oocytes into a microwell plate (Nunc, 10 &#181;l well volume). This should be done carefully, using a 200-&#181;l pipettor with a pipette tip that has been cut at the end to allow undisrupted suction of the oocytes.</li>
</ol>
Part 2: Microinjection of Xenopus oocytes
<ol>
	<li>Prepare the import substrate to be microinjected by adding a small amount of 1% bromphenol blue, to aid in the visualization of microinjection. For example, we add 1 &#181;l of bromphenol blue to 10 &#181;l of substrate.</li>
	<li>Place a small strip of Parafilm on a 100-mm diameter Petri dish, and dispense a 5-&#181;l drop of the solution to be injected on the Parafilm strip.</li>
	<li>Fill a previously calibrated injection needle (obtained by pulling a 6.6-&#181;l micropipette Drummond with the Inject+Matic Puller) with the solution to be injected. For this, the microinjector is set on aspiration mode. To calibrate the injection needle make dot marks on the needle every 0.5 mm, which correspond to a volume of 50 nl.</li>
	<li>For cytoplasmic injection, insert the tip of the needle into an oocyte in the vegetal hemisphere, very close to the animal hemisphere, at an approximately 45-degree angle. Turn the microinjector setting to microinject, and microinject each oocyte with 50 nl of import substrate. The dot marks on the needle are used to monitor the amount of substrate that has been injected.</li>
	<li>Transfer the injected oocytes to a small (35-mm diameter) Petri dish filled with MBS, and incubate at room temperature for the desired amount of time (time points are chosen so as to allow observation of the import substrate associated with NPCs; this occurs between 10 and 30 minutes for proteins and for viruses that are actively transported toward the NPC; this time also depends on the site of injection and the size of the protein/virus).</li>
	<li>When the incubation is complete, transfer oocytes to a 4-ml glass vial containing 2% glutaraldehyde in MBS, and fix overnight at 4 &#176;C.</li>
</ol>
Part 3: Dissection of Xenopus oocytes
<ol>
	<li>The next day, wash the oocytes 3 times with MBS.</li>
	<li>Transfer oocytes to a small Petri dish filled with low salt buffer (LSB: 1 mM KCl, 0.5 mM MgCl2, 10 mM HEPES, pH 7.5). Using a dissecting microscope and dissecting tweezers, remove the vegetal pole from each oocyte. It is useful to stabilize the oocyte being dissected with one pair of tweezers, while the other pair is used like scissors to remove the vegetal pole. At this point it is possible to evaluate the success of the microinjection by the presence of a blue tinge in the cytosol. The protocol is continued only for the oocytes that were microinjected successfully. In addition, if the samples are to be prepared for EM, this dissection step makes it much easier to find the nucleus when trimming and sectioning the samples.</li>
	<li>Fix the dissected oocytes again with 2% glutaraldehyde in LSB for one hour at room temperature.</li>
	<li>After fixation, wash the dissected oocytes three times with LSB.</li>
</ol>
Part 4: Preparation of Injected Oocytes for Embedding and Thin-Sectioning EM
<ol>
	<li>Transfer the dissected oocytes to a depression slide. Aspirate as much of the liquid as possible, and then quickly (so that they do not dry out) cover the dissected oocytes with 2% low-melting agarose. While the agarose is still soft, use a pipette tip to separate the oocytes from each other, and to ensure that each oocyte is face up or down (not sideways).</li>
	<li>Allow the agarose to solidify for about 10 minutes. Once the agarose solidifies, use a razor blade to cut the agarose into small pieces, each containing one dissected oocyte.</li>
	<li>Place the agarose-embedded oocytes in a 4-ml glass vial, and post-fix them with 1% OsO4 in LSB for one hour at room temperature.</li>
	<li>Wash the sample three times in LSB. If necessary, store the sample at 4 &#176;C overnight and continue the protocol the next day.</li>
	<li>Sequentially dehydrate the samples in an ascending series of alcohol (50, 70, and 90% ethanol for 20 min each). This is followed by two changes in 100% ethanol for 15 min each. Finally dehydrate samples with 100% acetone for 15 min.</li>
	<li>Infiltrate samples with a 1:1 mixture of Epon (Fluka) and acetone for one hour, followed by infiltration with a 2:1 mixture of Epon and acetone for two hours, and finally in pure Epon for at least six hours.</li>
	<li>Place samples in flat embedding molds filled with fresh pure Epon. The oocytes are oriented in such a way that the side of the nucleus closer to the injection site &#8211; in this case the side of the oocytes that has been dissected &#8211; will be sectioned first. This step optimizes the chance of visualizing import of the chosen substrate.</li>
	<li>Finally, polymerize the Epon for two days at 60 &#176;C.</li>
	<li>In order to visualize the samples, the blocks are trimmed and then sectioned using an ultramicrotome. Sections are transferred onto EM grids, stained using standard procedure, and visualized with a transmission electron microscope. We will not show this part of the protocol as it is standard EM procedure.</li>
</ol>
Part 5: Representative Results:
If the protocol has been successful, then the nuclear envelope (NE) and NPCs should be clearly visible in EM micrographs. Depending on the substrate injected and the amount of time between injection and fixation, the substrate should be visible at the cytoplasmic face of the NPC, in the NPC, or at the nuclear face of the NPC.
Figure 1 shows a NE cross-section with adjacent cytoplasm (c) and nucleus (n) from a Xenopus oocyte that has been injected with capsids of the baculovirus AcMNPV, incubated at 4&#176;C for four hours, and processed for embedding and thin section EM as described. Arrowheads point to NPCs. A capsid docking at the cytoplasmic face of a NPC is indicated by a white arrow.
In contrast, Figure 2 shows a NE cross-section with adjacent cytoplasm (c) and nucleus (n) from a Xenopus oocyte that has been injected with the parvovirus Minute Virus of Mice (MVM), incubated at room temperature for four hours, and processed for embedding and thin section EM as described. Using this technique, we have found that MVM induces disruptions of the NE (Cohen and Pant&#233;, 2005). Brackets indicate breaks in the NE. Arrowheads point to NPCs. Putative MVM capsids associated with the NE are indicated by white arrows.
<img alt="Figure 1" src="/files/ftp_upload/1106/1106fig1.jpg" />
Figure 1: Electron micrograph of a NE cross-section with adjacent cytoplasm (c) and nucleus (n) from a Xenopus oocyte that has been injected with capsids of the baculovirus AcMNPV, incubated at 4&#176;C for four hours, and processed for embedding and thin section EM as described. Arrowheads point to NPCs. Capsids are indicated by white arrows. Scale bar: 100 nm.
<img alt="Figure 2" src="/files/ftp_upload/1106/1106fig2.jpg" />
Figure 2: Electron micrograph of a NE cross-section with adjacent cytoplasm (c) and nucleus (n) from a Xenopus oocyte that has been injected with the parvovirus Minute Virus of Mice (MVM), incubated at room temperature for four hours, and processed for embedding and thin section EM as described. Arrowheads point to NPCs. Brackets indicate breaks in the NE. Putative MVM capsids associated with the NE are indicated by white arrows. Scale bar: 100 nm.

<ol>
	<li>Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+intact+Xenopus+oocytes+in+nucleocytoplasmic+transport+studies.">Use of intact Xenopus oocytes in nucleocytoplasmic transport studies.</a> Methods Mol. Biol. 322, 301-314 (2006).</li><li>Pant&#233;, N., Kann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+pore+complex+is+able+to+transport+macromolecules+with+diameters+of+about+39+nm.">Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm.</a> Mol. Biol. Cell. 13 (2), 425-434 (2002).</li><li>Rabe, B., Vlachou, A., Pant&#233;, N., Helenius, N., Kann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+hepatitis+B+virus+capsids+and+release+of+the+viral+genome.">Nuclear import of hepatitis B virus capsids and release of the viral genome.</a> Proc. Natl. Acad. Sci. U.S.A. 100 (17), 9849-9854 (2003).</li><li>Cohen, S., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pushing+the+Envelope:+Microinjection+of+MVM+into+Xenopus+oocytes+causes+damage+to+the+nuclear+envelope.">Pushing the Envelope: Microinjection of MVM into Xenopus oocytes causes damage to the nuclear envelope.</a> J. Gen. Virol. 86 (12), 3243-3252 (2005).</li><li>Pant&#233;, N., Jarmolowski, A., Izaurralde, E., Sauder, U., Baschong, W., Mattaj, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+nuclear+export+of+different+classes+of+RNA+by+electron+microscopy.">Visualizing nuclear export of different classes of RNA by electron microscopy.</a> RNA. 3, 498-513 (1997).</li></ol>

Microinjection of Xenopus oocytes combined with thin-sectioning EM is a highly effective tool for studying nucleocytoplasmic transport. This system has been used to map distinct steps of import through the NPC, for example interaction of a nuclear import substrate with structural components of the NPC such as the cytoplasmic filaments and nuclear basket (reviewed by Pant&eacute;, 2006). It has also been used to study the nuclear import of nuclear-replicating viruses (Pant&eacute; and Kann, 2002; Rabe et al.<...

microinjection of xenopus laevis oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Watch this Scientific Journal Video about Microinjection of Xenopus Laevis Oocytes at JoVE.com

Microinjection of Xenopus Laevis Oocytes

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

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22.9K Views.  University of British Columbia - UBC. כאן אנו מדגימים microinjection cytoplasmic של Xenopus laevis ביציות עם מצע לייבא גרעיני, כמו גם הכנה של ביציות מוזרק להדמיה באמצעות מיקרוסקופ אלקטרונים חתך דק.

Video: Microinjection of Xenopus Laevis Oocytes

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

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.

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

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...