We thank Dartmouth College&#39;s Center for Comparative Medicine and Research for providing daily husbandry for the animals used in this protocol. We also thank Leah Jacob and Adwaita Bose for their help in testing the protocol and photographing animals. Lastly, we thank Ann Miller&#39;s lab for training in the practice of tattooing. This work was supported by NIH grant R00 GM147826 to J.L.

All animal procedures described were approved by Dartmouth College&#39;s Institutional Animal Care and Use Committee.
1. Equipment setup
NOTE: A workflow for the procedure and an example bench setup are included (Figure 1).
<ol>
	<li>Connect the tattoo gun and foot pedal to the power supply. Position the foot pedal underneath the working surface.</li>
	<li>Assembling the tattoo gun
	<ol>
		<li>Using an Allen wrench or hex screwdriver, loosen the screws in the grip (Figure 2A, encircled in red).</li>
		<li>Insert the plastic tip all the way into the grip. Tighten the screw to hold it in place.</li>
		<li>Insert the metal tube in the back of the grip (Figure 2A). This will be adjusted, so insert it to half its length. Tighten the screw to hold it into place.</li>
		<li>Insert a tattoo needle through the back of the grip so that it sits comfortably in the plastic tip at the front of the grip (Figure 2B).</li>
		<li>Slide the back of the needle and metal tube through the tube clamp. Adjust the metal tube so that the needle fits comfortably in the plastic tip (Figure 2B). Hand-tighten the tube clamp until the tube is unable to move (Figure 2C).</li>
		<li>Remove the black O-ring and hook the needle onto the rotor arm; replace the O-ring to secure the needle into place (Figure 2C).</li>
	</ol>
	</li>
	<li>Set the voltage on the power supply so that there is enough power to pierce the skin but not so powerful that it is difficult to control. Once the power supply is plugged in and connected, press the foot pedal to make sure the machine is working. Take care not to point the needle at yourself or other personnel. 
	NOTE: The tattoo machine used in this protocol works best between 6.0 and 9.0 V, but this may vary between tattoo machines and voltage supplies and should be determined empirically.</li>
	<li>Fill the cap of a 1.5 mL microcentrifuge tube or a provided plastic inkpot with black tattoo ink to about three-quarters full.</li>
</ol>
2. Anesthesia
<ol>
	<li>Prepare anesthesia in a tank large enough to submerge one adult female X. laevis frog.
	<ol>
		<li>Using frog-safe (chlorine-free) water, add tricaine to the final concentration of 1.5 g/L and sodium bicarbonate to the final concentration of 3.5 g/L. Mix to dissolve. The pH of this solution is 7.15. 
		CAUTION: Tricaine is an irritant.</li>
	</ol>
	</li>
	<li>Submerge one frog in the anesthesia tank. Ensure that the frog stays submerged in the tricaine solution until it is anesthetized (7-8 min).
	<ol>
		<li>To check if the frog is fully anesthetized, pick it up, hold it upside down, and give it a firm squeeze on one foot. If the frog does not flinch or respond, it can be tattooed.</li>
		<li>If frogs consistently take more than 10 min to be anesthetized, prepare fresh tricaine solution buffered with sodium bicarbonate. 
		NOTE: Frogs should not spend more than 30 min in tricaine solution15.</li>
	</ol>
	</li>
</ol>
3. Tattooing
NOTE: Before tattooing a live animal, it can be useful to practice on a piece of fruit with a firm peel (such as a lemon or banana).
<ol>
	<li>Prepare a recovery tank for the frogs before tattooing. Fill a tank with 10-15 L of fresh frog-safe water and add a Styrofoam island. This will provide a surface for the frog to wake up without drowning. 
	NOTE: A Styrofoam island can be made using the lid of a Styrofoam cold shipping container placed inside a zip-top bag.</li>
	<li>Place the anesthetized frog onto its back on a dry paper towel or bench paper (Figure 3A). Use unbleached (brown) paper towels for all work involving frogs.</li>
	<li>Using a dry lint-free wipe, wipe away the water and mucus from the frog&#39;s chest.</li>
	<li>Find the sternum in the center of the frog&#39;s chest with fingers and, using the non-dominant hand, hold the skin taut.</li>
	<li>Marking the frog
	<ol>
		<li>Hold the assembled tattoo gun vertically relative to the working surface and dip the needle into the ink (Figure 3B).</li>
		<li>Keeping the assembled tattoo gun vertical, press the tip of the needle onto the frog&#39;s skin before pressing the foot pedal. Draw lines onto the frog&#39;s skin while applying even pressure. 
		NOTE: Some minor bleeding or redness is normal, as is minor skin sloughing.</li>
		<li>If the needle is getting caught on the frog&#39;s skin, clear excess ink or skin from the needle with a wipe. Excess ink is normal during tattooing.</li>
		<li>If the frog&#39;s chest is covered in too much ink to clearly see the area being marked (Figure 3C), clear excess ink with frog-safe water and a wipe (Figure 3C&#39;). Then, wick away moisture with a dry wipe and continue inking. 
		NOTE: A squeeze bottle of frog-safe water can be prepared before tattooing.</li>
		<li>Continue to ink the same area until dark, legible numbers remain after wiping away excess ink (Figure 3D). Instead of drawing the entire marking or number in one stroke, repeatedly make smaller strokes, especially for curves.</li>
	</ol>
	</li>
</ol>
4. Recovery
<ol>
	<li>Wet a paper towel with frog-safe water and place it flat on top of the Styrofoam island (Figure 4A).</li>
	<li>Returning frogs to standing tanks
	<ol>
		<li>Lay the frog belly-down on the towel facing the water (Figure 4B). Fold half of the paper towel over the back half of the frog and, using cupped hands, wet the top of the frog. Once a frog emerges from anesthesia (about 1 h after tattooing), they will enter water independently and swim normally.</li>
		<li>Return frogs to long-term housing after 24 h, once the tattoo has fully healed.</li>
	</ol>
	</li>
</ol>
5. Cleaning and equipment maintenance
NOTE: Although needles can be reused for multiple frogs in the same sitting, best practice is to replace or sterilize needles between tattooing frogs.
<ol>
	<li>Reusing and autoclaving tattooing needles
	<ol>
		<li>Rinse needles in 100% isopropanol and deionized water to loosen the ink and scrub off the remaining ink that has not dissolved.</li>
		<li>After scrubbing, place the needles into a metal autoclaving case or wrap them with aluminum foil and autoclave for 30 min on a dry cycle.</li>
	</ol>
	</li>
	<li>If there is ink on pieces of the tattoo machine or other equipment, wipe it with 100% ethanol and a paper towel.</li>
	<li>The anesthetic solution has a final tricaine concentration of approximately 0.5%. Dispose of this according to institutional hazardous waste management guidelines.</li>
	<li>Equipment storage and maintenance
	<ol>
		<li>If possible, store the tattoo machine in a dry room. Excess moisture will damage the electric components and reduce the longevity of the machine.</li>
		<li>To keep the tattoo machine in good working order, check the bearing on the rotor (Figure 2C) to make sure it has not come loose from vibrations22. This can be tightened with an Allen wrench or hex screwdriver.</li>
		<li>About once a month, lubricate the bearing on the rotor arm using a thick lubricating grease22.</li>
		<li>On some tattoo machines, the rotor arm can get stuck and will not vibrate. To correct this, twist the rotor arm or compress the spring so that the needle is in the lowered position (Figure 2C).</li>
	</ol>
	</li>
</ol>

Optimizing Amphibian Marking: A Guide to Tattooing Xenopus

<ol>
	<li>Hubrecht, R. C., Carter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+3Rs+and+humane+experimental+technique:+Implementing+change.">The 3Rs and humane experimental technique: Implementing change.</a> Animals (Basel). 9 (10), 754 (2019).</li><li>Elkan, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus+pregnancy+test.">The Xenopus pregnancy test.</a> British Med J. 2, 1253-1256 (1938).</li><li>Vize, P. D., Zorn, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+genomic+data+and+browser+resources.">Xenopus genomic data and browser resources.</a> Dev Biol. 426 (2), 194-199 (2017).</li><li>Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xenopus: A Laboratory Manual. , (2023).</li><li>Borodinsky, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+as+a+model+organism+for+the+study+of+spinal+cord+formation,+development,+function+and+regeneration.">Xenopus laevis as a model organism for the study of spinal cord formation, development, function and regeneration.</a> Front Neural Circuits. 11, 90 (2017).</li><li>Hoppler, S., Conlon, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus:+Experimental+access+to+cardiovascular+development,+regeneration+discovery,+and+cardiovascular+heart-defect+modeling.">Xenopus: Experimental access to cardiovascular development, regeneration discovery, and cardiovascular heart-defect modeling.</a> Cold Spring Harb Perspect Biol. 12 (6), a037200 (2020).</li><li>Fisher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenbase:+key+features+and+resources+of+the+Xenopus+model+organism+knowledgebase.">Xenbase: key features and resources of the Xenopus model organism knowledgebase.</a> Genetics. 224 (1), 018 (2023).</li><li>Slater, P. G., Hayrapetian, L., Lowery, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+as+a+model+system+to+study+cytoskeletal+dynamics+during+axon+pathfinding.">Xenopus laevis as a model system to study cytoskeletal dynamics during axon pathfinding.</a> Genesis. 55 (1-2), 22994 (2017).</li><li>Bermudez, J. G., Chen, H., Einstein, L. C., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+biology+of+cell+boundary+conditions+through+confinement+of+Xenopus+cell-free+cytoplasmic+extracts.">Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.</a> Genesis. 55 (1-2), 23013 (2017).</li><li>Stooke-Vaughan, G. A., Davidson, L. A., Woolner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+as+a+model+for+studies+in+mechanical+stress+and+cell+division.">Xenopus as a model for studies in mechanical stress and cell division.</a> Genesis. 55 (1-2), 23004 (2017).</li><li>Blackburn, A. T. M., Miller, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+congenital+kidney+diseases+in+Xenopus+laevis.">Modeling congenital kidney diseases in Xenopus laevis.</a> Dis Model Mech. 12 (4), 038604 (2019).</li><li>Tandon, P., Conlon, F., Furlow, J. D., Horb, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+toolkit+in+Xenopus:+Approaches+and+opportunities+for+human+disease+modeling.">Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling.</a> Dev Biol. 426 (2), 325-335 (2017).</li><li>Wheeler, G. N., Br&#228;ndli, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+vertebrate+models+for+chemical+genetics+and+drug+discovery+screens:+Lessons+from+zebrafish+and+Xenopus.">Simple vertebrate models for chemical genetics and drug discovery screens: Lessons from zebrafish and Xenopus.</a> Dev Dyn. 236 (6), 1287-1308 (2009).</li><li>Carron, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+origin+of+Hox13-dependent+skin+appendages+in+amphibians.">Evolutionary origin of Hox13-dependent skin appendages in amphibians.</a> Nat Commun. 15 (1), 2328 (2024).</li><li>Schultz, T. W., Dawson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Housing+and+husbandry+of+Xenopus+for+oocyte+production.">Housing and husbandry of Xenopus for oocyte production.</a> Lab Animal. 32 (2), 34-39 (2003).</li><li>Cadillac, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Animal identification systems used for mice. , (2006).</li><li>Donnelly, M. A., Guyer, C., Juterbock, J. E., Alford, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+marking+amphibians.">Techniques for marking amphibians.</a> Meas Monitor Biol Div: Std Meth Amphibians. , 277-284 (1994).</li><li>Hutchens, S. J., Deperno, C. S., Matthews, C. E., Pollock, K. H., Woodward, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible+implant+fluorescent+marker:+A+reliable+marking+alternative+for+snakes.">Visible implant fluorescent marker: A reliable marking alternative for snakes.</a> Herpetol Rev. 39 (3), 301-303 (2008).</li><li>Hoogstraten-Miller, S., Dunham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+identification+methods+for+African+clawed+frogs+(Xenopus+laevis).">Practical identification methods for African clawed frogs (Xenopus laevis).</a> Lab Animal. 26 (7), 36-38 (1997).</li><li>Loopstra, J. A., Zwart, P., Verhoeff-de Fremery, R., Vervoordeldonk, F. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marking+of+African+clawed+toads+(Xenopus+laevis).+Improvement+of+a+skin+autograft+technique.">Marking of African clawed toads (Xenopus laevis). Improvement of a skin autograft technique.</a> New Dev Biosci: Their Implicat Lab Animal Sci. , 311-316 (1988).</li><li>Rodel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis.">Xenopus tropicalis.</a> AmphibiaWeb. , (2001).</li><li>Worldwide Tattoo Supply. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stealth+2+Rotary+Tattoo+Machine+Intro+&amp;+Maintenance.">Stealth 2 Rotary Tattoo Machine Intro &amp; Maintenance.</a> Worldwide Tattoo Supply. , (2015).</li><li>Khan, S. U., Mufti, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hot+history+and+cold+future+of+brands.">The hot history and cold future of brands.</a> J Manager Sci. 1 (1), 75-84 (2007).</li><li>Catlett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+About+Lip+Tattoo:+what+to+Expect,+Cost,+Risks,+Touch-ups+&amp;+More.">All About Lip Tattoo: what to Expect, Cost, Risks, Touch-ups &amp; More.</a> Healthline. , (2019).</li></ol>

The authors declare no competing interests.

Tattooing humans is an art form dating back thousands of years, and for as long as humans have tattooed themselves, they have also tattooed or branded animals23. The equipment and techniques for marking animals, particularly mammals, are well-established, well-documented, and widely accessible. While marking animals was originally for distinguishing livestock and theft deterrents23, its role in biomedical research has become just as important.
A ...

Animal models are useful tools for investigating questions pertaining to human health. In practice, biomedical research using animal models requires careful organization and maintenance of a healthy animal colony. Best practices for ethical animal handling and husbandry aim to reduce the number of animals needed for experimentation and refine practices to ensure animal welfare1. The clawed frog genus, including Xenopus laevis (X. laevis; African clawed frog) and Xenopus tropicalis (X. tropicalis; Western clawed frog), have been used in biomedical research since the 1930s when X. laevis were used by South African doctors to conduct the first pregnancy tests2. While modern pregnancy tests no longer require frogs, the role of Xenopus research persists. Advantages of using Xenopus for biomedical research include their well-annotated genomes3, year-round inducible ovulation of large clutches of eggs4, and externally laid eggs amenable to in vitro fertilization. These features make them a useful asset for vertebrate embryology and development5,6,7, basic molecular and cellular biology7,8,9,10, and for modeling human disease7,11,12,13.
Reliable methods for tracking individual Xenopus animals are essential for recording biological replicates and improving rigor and reproducibility in research. As Xenopus are frequently housed in groups, animal marking allows researchers to easily track individual animals4. Maintaining an accurate record of animals can save time and resources and improve the ability to track animals&#39; health. For example, individual identification of animals can improve organization workflows for generating transgenic Xenopus lines, as this requires multiple generations of frogs with specific genotypes verified by sequencing14, which requires organization and individual identification of animals. This is particularly true when these mutations lack easily discernible adult phenotypes. Similarly, the use of Xenopus oocytes and embryos to study basic cellular and developmental biology benefits from tracking individual animals. After inducing ovulation, animals need to rest for a minimum of 3 months to prevent health complications such as hyper-ovulation syndrome15. Individual identification methods ensure that animals are not induced to ovulate too frequently.
Marking and tracking animals also enables lab personnel to track animals&#39; health concerns. Animals of the same genotype falling ill can indicate excessive inbreeding or unanticipated health concerns associated with the transgene. Similarly, animals falling ill after recent ovulation can indicate issues with reagents, materials, or techniques. Tracking animals and their health enables lab personnel and veterinarians to follow up when concerns resurface and take preventative measures to prevent future illness. In mammals, there are numerous identification methods. Permanent methods for mice include ear punching, ear tags, tattooing, and subcutaneous microchips16. These can clearly and reliably differentiate animals within a colony or cage and can be easily administered by laboratory personnel. Methods such as ear punching are minimally invasive, require only one piece of specialized equipment, and work for animals of most ages. While these systems are straightforward and useful for mice, their use in frogs presents a unique set of challenges. Frogs and other amphibians lack a pinna (external ear structure). Some researchers have attached tags to the animal&#39;s jaw, toe, or hind limb17,18. This approach resulted in various problems: jaw tags caused irritation, and agitated frogs attempted to pull off tags with their forelimbs17. Toe tags pierced the webbing between toes, impairing movement and carrying the risk of becoming lost. As such, amphibians require their own methods for identification. Historically, toe-clipping has also been used to mark amphibians17,19. The toe is clipped with a sharp pair of scissors, and the animal can be identified by the length of toes on the forefeet and hind feet or by the angle at which the toe has grown back (in salamanders). However, this method poses the ethical concern that toe clipping may impair the animal&#39;s movement17. In addition, this can cause bleeding and introduce a risk of infection. Another established marking system is skin autografts, in which skin is taken from one part of the frog and surgically attached to another part. For example, a method is described for marking a frog&#39;s back or shoulder using a light-colored skin graft from its chest20. Skin grafts also come with limitations and risks: the procedure is invasive and introduces the risk of Aeromonas hydrophila infection, or red leg, a potentially fatal affliction; complete healing of the autograft takes up to 6 weeks; and with the methods described, only 6 frogs can be housed together because of limited places to put an autograft20.
Less invasive marking approaches include glass beads and transponder chips17,19. In the glass bead method, glass beads are threaded onto a small suture and sewn into the frog&#39;s skin. This provides greater variability than skin autografts, with at least 60 distinctive color combinations. There is, however, a risk that the suture can come out and result in the beads being lost. Alternatively, a microchip transponder can be implanted under the skin in the frog&#39;s dorsal lymph sac. This is considered the most permanent marking method and enables a potentially infinite number of animals to be individually identified and cataloged. However, this is also the most expensive method, as individual microchips are expensive, and a large colony would be costly to mark. Microchips also require a special scanner to read19. One common approach for Xenopus identification is referring to animals&#39; natural coloring and patterning. This works especially well for frogs such as X. laevis, which have distinct patterns that remain throughout adulthood. However, these patterns can change over time with stress, and coloring can appear different when frogs are moved between transparent and tinted containers15. Additionally, this identification method is less useful for X. tropicalis, which has less distinct marking patterns compared to X. laevis, or for albino animals, which have no color markings21. Even for species with distinct markings, lab personnel can interpret the placement and size of markings differently, which can cause errors in identification. Because of this, photographing animals is most reliable in conjunction with an additional identification method. Therefore, we seek to mark and identify Xenopus animals using a technique that is easily discernable, permanent, and minimally invasive.
There are limited published resources describing methods for tattooing amphibians. Tattooing has been described alongside other branding techniques, including heat brands, silver nitrate brands, and freeze brands17. In the same resource, tattooing was done by drawing numerals with a 27G hypodermic needle, and the process was noted not to cause infection, in contrast to the other branding techniques, which used a wire shaped into a numeral or other mark. In another source, an electric tattooing machine (described as a vibrating needle) was used to mark frogs, but little detail was provided on the technique17,19. The authors warn that by disturbing the frog&#39;s protective slime layer, this procedure increases the risk of red leg. While there is no marking or identification method that is both completely noninvasive (such as photographs) and permanent (such as microchips), tattooing provides an effective compromise. Tattooing is relatively straightforward compared to other techniques, such as skin autografts. Additional benefits include a smaller learning curve and relatively inexpensive equipment. Tattooing aquatic amphibians comes with certain challenges, which can intimidate researchers and impair successful animal marking. This paper aims to provide researchers with well-documented methods for tattooing adult Xenopus with a rotary tattoo machine.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 needle round liners</td>
			<td>Worldwide Tattoo Supply</td>
			<td>1203RLB</td>
			<td>Packaged sterile</td>
		</tr>
		<tr>
			<td>5 Needle Round Disposable ULTRA</td>
			<td>Worldwide Tattoo Supply</td>
			<td>HTIPRS5-U</td>
			<td>Packaged sterile</td>
		</tr>
		<tr>
			<td>5 needle round liners</td>
			<td>Worldwide Tattoo Supply</td>
			<td>1205RLB</td>
			<td>Packaged sterile</td>
		</tr>
		<tr>
			<td>7 needle round liners</td>
			<td>Worldwide Tattoo Supply</td>
			<td>1207RLB</td>
			<td>Packaged sterile</td>
		</tr>
		<tr>
			<td>Clip Cord</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Foot pedal</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inkpots</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, delicate task wipes</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>RCA Connection</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scream Ink Pitch Black, 1oz</td>
			<td>Worldwide Tattoo Supply</td>
			<td>SI101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel grips</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stealth 2.0 Rotary Tattoo Machine</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stealth 2.0 Rotary Tattoo Machine Box Set</td>
			<td>Worldwide Tattoo Supply</td>
			<td>STEALTH2-SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam island</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This is the lid of a styrofoam cold shipping container</td>
		</tr>
		<tr>
			<td>Tricaine (ethyl 3-aminobenzonate methanesulfate)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>CAUTION: IRRITANT</td>
		</tr>
		<tr>
			<td>Unbleached paper towels</td>
			<td>Grainger</td>
			<td>2U229</td>
			<td>Paper towels MUST be unbleached, bleach is toxic to amphibians</td>
		</tr>
		<tr>
			<td>Voltage Supply</td>
			<td>Worldwide Tattoo Supply</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash bottle (with frog-safe water)</td>
			<td>Fisher Scientific</td>
			<td>FB0340923T</td>
			<td>Frog safe water is dechlorinated, pH 7.0-8.5, conductivity 1200-1800 uS</td>
		</tr>
		<tr>
			<td>X. laevis adult female</td>
			<td>Xenopus1</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Zip-top plastic bag</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>This bag should be large enough to hold the styrofoam island</td>
		</tr>
	</tbody>
</table>

methods for tattooing xenopus laevis with a rotary tattoo machine

Animal models expand the scope of biomedical research, furthering our understanding of developmental, molecular, and cellular biology and enabling researchers to model human disease. Recording and tracking individual animals allows researchers to reduce the number of animals required for study and refine practices to improve animal wellbeing. Several well-documented methods exist for marking and tracking mammals, including ear punching and ear tags. However, methods for marking aquatic amphibian species are limited, with the existing resources being outdated, ineffective, or prohibitively costly. In this manuscript, we outline methods and best practices for marking Xenopus laevis with a rotary tattoo machine. Proper tattooing results in high-quality tattoos, making individuals easily distinguishable for researchers and posing minimal risk to animals' health. We also highlight the causes of poor-quality tattoos, which can result in tattoos that fade quickly and cause unnecessary harm to animals. This approach allows researchers and veterinarians to mark amphibians, enabling them to track biological replicates and transgenic lines and to keep accurate records of animal health.

High-quality tattoos will have dark, legible strokes on the frog&#39;s chest and can be clearly differentiated from several feet away (Figure 5A). In general, larger numbers and markings are better for readability, but longer names and numbers can be made smaller to fit comfortably on the frog&#39;s chest. Tattoo longevity is more difficult to judge, but high-quality frog tattoos should remain dark and legible for at least 3-6 months (Figure 5D-D&#39;</s...

Author Spotlight: Refining Xenopus laevis Marking Techniques for Biomedical Studies

Here, we describe methods for tattooing adult Xenopus laevis (African clawed frog) with a rotary tattoo machine. Proper tattooing results in dark, easily legible numerals that last for several months and make animals easily distinguishable for research and recordkeeping purposes.

Methods for Tattooing Xenopus laevis with a Rotary Tattoo Machine

Animal models expand the scope of biomedical research, furthering our understanding of developmental, molecular, and cellular biology and enabling ...

methods-for-tattooing-xenopus-laevis-with-a-rotary-tattoo-machine

Research

JoVE Journal

Biology

556 Views.  Dartmouth College. Here, we describe methods for tattooing adult Xenopus laevis (African clawed frog) with a rotary tattoo machine. Proper tattooing results in dark, easily legible numerals that last for several months and make animals easily distinguishable for research and recordkeeping purposes.

Video: Author Spotlight: Refining Xenopus laevis Marking Techniques for Biomedical Studies

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

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 ...

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.

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 ...

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 ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

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. ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.