Table 1. Comparison between tail vein and retro-orbital injection.

		Tail vein injection	Retro-orbital injection
1	Injection site	Lateral caudal vein	Retro-orbital plexus
2	Age	at least 6 weeks old	Suitable for all ages
3	Restraining device	Required	Not required
4	Anesthesia
	* newborn (P0-P1)	Not applicable	cryoanesthesia
	* neonates (≥1 week old)	Not applicable	Inhalant anesthesia (1.5% isoflurane)
	* juvenile and adult	Not required	Inhalant anesthesia (2.5% isoflurane)
5	Injection volume
	* newborn (P0-P1)	Not applicable	~ 10 μl (per eye)
	* neonates (P14 to P21)	Not applicable	Up to 50 μl (per eye)
	* juvenile and adult	Up to 200 μl	Up to 200 μl (per eye)
6	Continuous injections	Yes	Yes
7	Stress level	High	Low
8	Vasodilation	Required (heat lamp or warm water)	Not required
9	Needle size	28 G	31G (for neonates)
			28G or smaller (for adult)
10	Disinfection	Swab the tail with 70% ethanol	Not required
11	Time required	Relatively longer*	Shorter
12	Visibility of the vein	Obvious in non-pigmented mouse	Not applicable
13	Other benefits	Long injection route†	-
14	Other side effects	Possible inflammation or necrosis at injection site; possible scarring or bruising of the tail if substance administered subcutaneously	Swelling adjacent to the injection site, proptosis, eye trauma
15	Other optional procedures	-	Topical ophthalmic analgesic (tetracaine, gentamicin ophthalmic)

*Longer time required due to multiple procedures involved - to restrain animal; waiting time for vasodilation; to clean the restraint device, apply pressure on injection site to stop bleeding before returning to the home cage. ^†Long injection route - paired lateral vein runs the length of both lateral aspect along the tail and hence, can accommodate multiple attempts of injection, starting from distal to the proximal end of the vein.