Patel JH et al. (2024), Protocol for tail vein injection in Xenopus tro...

XB-ART-60586

STAR Protoc 2024 Feb 15;51:102895. doi: 10.1016/j.xpro.2024.102895.

Show Gene links Show Anatomy links

Protocol for tail vein injection in Xenopus tropicalis tadpoles.

Patel JH , Angell Swearer A , Kakebeen AD , Loh LR , Wills AE .

???displayArticle.abstract???
Functional studies in post-embryonic Xenopus tadpoles are challenging because embryonic perturbations often lead to developmental consequences, such as lethality. Here, we describe a high-throughput protocol for tail vein injection to introduce fluorescent tracers into tadpoles, which we have previously used to effectively inject morpholinos and molecular antagonists. We describe steps for safely positioning tadpoles onto agarose double-coated plates, draining media, injecting into the ventral tail vein, rehydrating plates, and sorting tadpoles by fluorescence with minimal injury for high-throughput experiments. For complete details on the use and execution of this protocol, please refer to Kakebeen et al.,1 Patel et al.,2 and Patel et al.,3.

???displayArticle.pubmedLink??? 38367232
???displayArticle.pmcLink??? PMC10882117
???displayArticle.link??? STAR Protoc
???displayArticle.grants??? [+]

Species referenced: Xenopus tropicalis

???attribute.lit??? ???displayArticles.show???

References [+] :

Aztekin, The myeloid lineage is required for the emergence of a regeneration-permissive environment following Xenopus tail amputation. 2020, Pubmed, Xenbase

	Graphical abstract
	Figure 1. Agarose coated lid next to an uncoated lid
	Figure 2. Microinjection rig setup with loaded capillary and tadpoles oriented for injection
	Figure 3. Plating of tadpoles for injection
	Figure 4. Expected results after injection with 2NBDG
	Figure 5. Expected results after injection with fluoro-Ruby
	Figure 6. Tadpoles after recovery from injection

	Figure 1. Agarose coated lid next to an uncoated lid
	Figure 2. Microinjection rig setup with loaded capillary and tadpoles oriented for injection
	Figure 3. Plating of tadpoles for injectionStart by plating a grid of tadpoles in individual droplets. Then, orient tadpoles with ventral sides in the same direction while removing most of the media. Anterior/posterior orientation does not matter. Finally, to prevent movement when injecting, remove as much of the media from each tadpole as possible. As an example, the top 4 rows of panel 3 show tadpoles ready for injection, compared to the bottom 4 rows. Top 4 rows are denoted by arrows.
	Figure 4. Expected results after injection with 2NBDGStage 41 tadpoles after injection with 4 nL of 5 mM 2NBDG. At 10 min, the signal is clearly seen in the vasculature between somites if injected into the tail vein. Signal is more uniformly distributed throughout most tissues by 3 h post injection. If misinjected, the fluorescent dye accumulates strongly at 10 min and 3 h post injection, seen by a much stronger signal in the notochord or somites. Each column is the same tadpole imaged at each time point. Arrows denote misinjection site.
	Figure 5. Expected results after injection with fluoro-RubyStage 41 tadpoles after injection with 4 nL of 500 μM fluoro-Ruby. At 10 min, the signal is seen in the vasculature between somites if injected into the tail vein. By 3 h post injection, the signal is largely accumulated in the kidney. If misinjected, the fluorescent dye accumulates strongly at 10 min and 3 h post injection. At 3 h post injection, some of the dextran will end up in the circulatory system and in the kidney, but the mis-targeted injection is still clearly visible. Arrows denote misinjection site.
	Figure 6. Tadpoles after recovery from injectionAfter completing this protocol, tadpoles should appear as on the left with minimal injury to the tail. If the tadpoles dry to the plate or injections take too long, the tail will be damaged as on the right. This most often leads to tearing of the fin tissue at the posterior of the tail.