Subcutaneous (SC or SQ) injection is the administration of a liquid substance into the subcutaneous space — the loose connective tissue layer directly beneath the skin and above the underlying muscle. In laboratory rodents, the subcutaneous space is especially accessible in the dorsal scruff (the loose skin over the upper back, between and just behind the shoulders), which is the most commonly used injection site. Compounds delivered SC are absorbed into the systemic circulation more slowly than IV or IP routes, as the drug must first diffuse through the loose tissue and into local capillaries and lymphatic vessels.
Subcutaneous injection is technically the simplest parenteral dosing route in rodents. The technique involves gently lifting a tent of loose skin at the injection site (commonly the dorsal scruff or dorsal flank in mice and rats), inserting the needle bevel-up at approximately 45° through the skin and into the space beneath, and injecting slowly. The formation of a small bleb or wheal under the skin confirms subcutaneous placement. The needle should not penetrate through to the underlying muscle — a needle that is too long or inserted too steeply may enter muscle tissue and effectively deliver an intramuscular injection.
The dorsal scruff (interscapular region) is preferred for several reasons: the skin is loose and easily tented, the subcutaneous space is relatively large, there are no major blood vessels or organs near the surface, and the site is accessible with one hand while restraining the animal. The dorsal flanks are also used, particularly when repeated SC injections are required and injection sites need to be rotated to avoid localised tissue irritation. In mice implanted with tumour models, the dorsal flank is a standard site for subcutaneous tumour implantation.
The subcutaneous route tolerates larger injection volumes than IV or intramuscular routes because the subcutaneous space can expand to accommodate a depot of fluid. For mice, up to 10 mL/kg body weight per injection site is the standard guideline for isotonic solutions; total doses exceeding 10 mL/kg should be divided across multiple sites. For rats, up to 5 mL/kg per site is the standard guideline. Non-isotonic, acidic, or alkaline formulations are more likely to cause local tissue irritation, pain, and necrosis at the injection site, so formulation pH and osmolality should be close to physiological where possible.
Common applications of the SC route include: slow-release drug formulations and depot preparations (where gradual release into circulation is desired), vaccine delivery, implantation of osmotic mini-pumps for continuous drug delivery, and tumour cell inoculation for xenograft or syngeneic cancer models. SC injection is also the preferred route when the compound causes significant vein irritation or when the compound's slow absorption profile is pharmacologically advantageous.
Key Points
- Inject into the dorsal scruff (interscapular region) or dorsal flank by tenting the skin; insert bevel-up at approximately 45°
- A visible bleb under the skin confirms subcutaneous placement
- Tolerates larger volumes than IV or IM routes: up to 10 mL/kg per site in mice for isotonic solutions
- Slower systemic absorption than IV or IP routes — used for depot and slow-release formulations
- Rotate injection sites for repeated SC dosing to avoid localised tissue irritation
Relevant Standards
- OECD Test Guidelines (specify SC as an acceptable dosing route for appropriate study types)
- ARRIVE Guidelines (reporting standards for animal research — require documentation of route, site, volume, and formulation)
- Institutional animal ethics committee (AEC in Australia; IACUC in North America) protocols govern approved volumes and technique
Related Terms
Frequently Asked Questions
What is the best injection site for subcutaneous injection in mice?
The dorsal scruff — the loose skin over the upper back, in the interscapular region between and just behind the shoulders — is the preferred site. Tent the skin by pinching it gently, insert the needle bevel-up at approximately 45° into the tent, and inject slowly. The skin in this region is loose enough to create a clear bleb when the injection is placed correctly. For repeated SC dosing, rotate between the scruff and both dorsal flanks to prevent localised tissue irritation.
How much can I inject subcutaneously in a mouse?
Up to 10 mL/kg body weight per injection site is the standard guideline for isotonic, non-irritant solutions in mice — for a 25 g mouse, that is approximately 0.25 mL per site. Total doses exceeding 10 mL/kg should be distributed across multiple sites to minimise discomfort. Always use the minimum effective volume, and confirm the approved volume with your institutional animal ethics committee.
How do I know if my subcutaneous injection was placed correctly?
A correctly placed SC injection produces a visible and palpable bleb (a small raised dome of fluid) under the skin at the injection site, which gradually disperses as the fluid is absorbed. If no bleb forms, the needle may have penetrated through to the underlying muscle — the injection was likely intramuscular rather than subcutaneous. If the bleb appears immediately but fluid is visible tracking under the skin away from the injection site, this is normal and indicates good subcutaneous placement with dispersal in the loose connective tissue.
What is the difference between subcutaneous and intramuscular injection in rodents?
Subcutaneous injection places the compound in the loose connective tissue layer directly under the skin. Intramuscular injection places the compound within skeletal muscle tissue. SC injections are absorbed more slowly, tolerate larger volumes, and carry less risk of nerve or vessel damage. IM injections are absorbed more rapidly due to the high vascularity of muscle, but the accessible muscle mass in mice and rats is small, limiting injectable volumes to approximately 0.05 mL per site in mice and 0.1–0.2 mL per site in rats.