Injections in mice are a day-to-day task for many labs. Deciding which type of injection to use depends on a number of factors. You have to weigh the pros and cons of various methods and choose what is best for you — and the mice. While not an exhaustive list, these can include what’s being injected, the type of experiment, the number of mice, the potential pain or discomfort of the mice, and restraint method. Many different substances can be injected, including cells, drugs, DNA, viral vectors, and viruses.
All researchers must go through the proper basic training prior to conducting mouse experiments. Some of these techniques are what we call “beginner”, or injections that are easy to pick up with minimal practice after initial mouse training. Other techniques require more skill and additional training, going from intermediate to advanced and finally, to expert.
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| Figure 1. Visual representation of common injection routes in mice. Image created with biorender.com. |
Needle techniques
Intravenous
Intravenous (IV) injections are most commonly administered through the tail vein — many researchers will simply refer to these as “tail vein injections.” However, they can also be given retro-orbitally, behind the eye. IV injections are used for a broad range of substances, as it circulate what is injected throughout the body. Tail vein injection may seem like the easier of the two, but it actually requires more technical skill to place the needle in the correct vein without collapsing it. Retro-orbital injections are also thought to be less stressful, because the mice are under anesthesia, but they are not recommended for tumorigenic compounds, as circulation is slower, and more institutional oversight is required.
Restraint: mechanical restraint or anesthesia
Location: tail vein or retro-orbitally
Gauge: 27–30
Volume: < 0.2 ml
Skill required: beginner (retro-orbital) to intermediate (tail vein)
Intraperitoneal
Intraperitoneal (IP) injections are given in the peritoneal cavity, or the space surrounding organs in the abdomen. They are a popular choice for administering different drugs and viral vectors. Like IV injections, intraperitoneal injections require a little bit of practice to ensure the proper injection site has been located and to avoid potential off-target pokes to nearby organs.
Restraint: scruffing
Location: peritoneal cavity
Gauge: 25–27
Volume: < 2–3 ml
Skill required: intermediate
Intramuscular
Intramuscular (IM) injections occur, as the name suggests, in the muscle. Common uses include administering vaccines, pain meds, or other drugs. Identifying the proper injection site is straightforward, as it targets large muscle groups (usually the thigh), though it is important to avoid injection into the sciatic nerve. Mice likely experience some muscle soreness after injection, like you might after getting a vaccine.
Restraint: mechanical restraint
Location: thigh
Gauge: 25–27
Volume: < 0.02–0.05 ml
Skill required: beginner
Subcutaneous
Subcutaneous (SQ or SC) injections are given between the skin and the muscles. The most common injection sites are flat and easily accessible, like on the back. Depending on the site and restraint method, subcutaneous injections take some practice to get the coordination down. Subcutaneous injections are most often used when injecting a cell suspension to create xenograft models.
Restraint: back scruffing or mechanical restraint
Location: back or hind legs
Gauge: 25–27
Volume: < 2–3 ml (~1 ml per site)
Skill required: beginner
Intradermal
Intradermal (ID) injections occur between the layers of the skin. While this could theoretically be performed anywhere on the body, somewhere large, flat, and easily accessible (like the back) is preferred. A common use of intradermal injections is studying inflammatory responses, as this method is useful for analyzing localized events. Intradermal injections require a bit of extra prep, as the injection site area must be shaved first.
Restraint: anesthesia
Location: between skin layers
Gauge: 26
Volume: < 0.05 ml
Skill required: advanced
Intrathecal
Intrathecal (IT) injections are probably the most advanced needle injection technique. Intrathecal injections are used when the substance being administered must be delivered to the central nervous system via cerebral spinal fluid. The injection site is the space in and around the vertebrae, so hitting the correct location requires a lot of skill and anatomical knowledge. Intrathecal injections are used for a wide array of purposes, but mainly for the delivery of various therapies and viral vectors. This injection sounds very disruptive, but surprisingly, it is considered minimally invasive.
Restraint: anesthesia
Location: spinal cord
Gauge: 30
Volume: up to 0.01 ml (10 µl)
Skill required: expert
No-needle techniques
The term “injection” is most often associated with needles, but it may surprise you that they don’t always require needles. If needles make you cringe or your experiment will allow you to avoid poking the mice, these “no needles” techniques may come in handy. These techniques are referred to as “instillation” or “gavage,” as they do not require any punctures.
Intratracheal instillation
Intratracheal instillation is used to deliver substances directly to the lungs. It is an advanced technique, as it requires intubation of the mice, which can be tricky given their small size. This technique is most often used for viral delivery, though drug delivery is also common. Due to the intubation, this procedure is considered quite invasive.
Restraint: anesthesia
Location: trachea
Gauge (tracheal tubing): 23–20
Volume: < 0.07 ml
Skill required: advanced
Intranasal instillation
Intranasal instillation is very similar to its intratracheal counterpart, as both procedures target the lungs. The main differences are that intranasal is easier, less invasive, and only requires a pipette. Since the substances are administered through the nose, they also pass through the upper airways and can target the airways before the lungs. This is most often used for delivery of viral vectors, though drug treatments are also possible. One additional requirement — patience, as you wait for the anesthetized mice to breathe in the liquid.
Restraint: anesthesia
Location: nose
Gauge: n/a
Volume: < 0.05 ml
Skill required: beginner
Oral gavage
Oral gavage delivers drugs that require ingestion, such as experimental therapies or pain relief, straight to the stomach. This technique involves administering medication through a feeding tube with a bulb on the end, which prevents accidental tears or punctures to the throat. The feeding tube can be flexible or metal, depending on researcher preferences. It sounds complicated but has a pretty low learning curve!
Restraint: scruffing
Location: throat
Gauge (bulb-tipped feeding tube): 18–20
Volume: up to 10 ml/kg
Skill required: intermediate
Your decision
Hopefully this post has provided a good introduction to common injection routes in mice! When considering what method you’d like to use, remember that the final decision will likely come down to experimental necessity, expertise available in your lab or organization, and approvals from the organization overseeing your work. Good luck with your in vivo experiments!
Table 1: Summary of common injection routes in mice.
| Injection Route | Restraint | Location | Gauge | Volume | Skill |
| Intravenous | Mechanical restrainer or anesthesia | Tail vein or retro-orbital | 27–30 | < 0.2 mL | Beginner (retro-orbital) to intermediate (tail vein) |
| Intraperitoneal | Scruffing | Peritoneal cavity | 25–27 | < 2–3 mL | Intermediate |
| Intramuscular | Mechanical restrainer | Thigh | 25–27 |
< 0.02–0.05 mL |
Beginner |
| Subcutaneous | Back scruffing or mechanical restrainer | Back or hind legs; between skin and muscle | 25–27 | < 2–3 mL (~1 mL per site) | Beginner |
| Intradermal | Anesthesia | Between skin layers | 26 | < 0.05 mL | Advanced |
| Intrathecal | Anesthesia | Spinal cord | 30 | up to 0.01 mL | Expert |
| Intratracheal | Anesthesia | Trachea | 20–23 (tubing) | < 0.07 mL | Advanced |
| Intranasal | Anesthesia | Nose | n/a | < 0.05 mL | Beginner |
| Oral gavage | Scruffing | Throat | 18–20 (bulb tipped) | up to 10 mL/kg | Intermediate |
Resources and references
Additional resources on the Addgene blog
Additional resources on Addgene.org
CRISPR Mammalian Expression plasmids
References
Prathipati, P., Rodriguez-Aguayo, C., Walton, B. L., Sood, A. K., Greaver, J., Janssen, C. F., & Lopez-Berestein, G. (2020). A retrospective analysis for different routes of administration in Mice-Percutaneous Retro-Orbital, jugular catheter, tail vein and femoral cut down injections. Journal of Biosciences and Medicines, 08(09), 131–141. https://doi.org/10.4236/jbm.2020.89011
Shimizu, S. S. (2004). Routes of administration. In H. Hedrich (Ed.), The Laboratory Mouse. Academic Press.
Turner, P. V., Brabb, T., Pekow, C., & Vasbinder, M. A. (2011). Administration of substances to laboratory animals: routes of administration and factors to consider. PubMed, 50(5), 600–613. https://pubmed.ncbi.nlm.nih.gov/22330705
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