How are rats frozen? - briefly
Rats are euthanized and then subjected to rapid cryogenic cooling, commonly by immersion in liquid nitrogen or placement in a -80 °C freezer, to achieve subzero preservation. The frozen specimens remain at these temperatures until they are processed for research.
How are rats frozen? - in detail
Freezing rats for experimental or preservation purposes follows a controlled protocol that minimizes tissue damage and ensures reproducibility. The procedure can be divided into three phases: pre‑freezing preparation, the freezing operation, and post‑freezing handling.
During preparation, the animal is anesthetized to prevent pain and reflex movements. Common agents include isoflurane or ketamine‑xylazine, administered according to weight‑based dosing guidelines. After loss of reflexes, the rat is positioned on a chilled metal platform to lower body temperature gradually, reducing the risk of thermal shock. Vital signs are monitored with a pulse oximeter or rectal probe to confirm stable physiological status before proceeding.
The freezing step employs one of two main techniques:
- Rapid cryogenic immersion – The animal is submerged in liquid nitrogen (‑196 °C) or a pre‑cooled isopentane bath (‑160 °C). Immersion time ranges from 30 seconds to 2 minutes, depending on body mass and desired ice crystal size. Immediate transfer to a pre‑cooled storage container prevents partial thawing.
- Controlled-rate freezer – The specimen is placed in a programmable freezer that lowers temperature at a defined slope (typically 1–3 °C per minute) from 4 °C down to –80 °C or lower. This method produces uniform ice formation and is preferred for preserving cellular integrity.
Throughout the cooling phase, temperature is recorded with thermocouples inserted into the core and peripheral sites to verify that the target temperature is reached without overshoot. Once the desired temperature is attained, the rat is sealed in a cryovial or wrapped in cryogenic foil, then stored in a liquid‑nitrogen dewar or ultra‑low‑temperature freezer (–80 °C) for long‑term preservation.
Post‑freezing handling includes labeling each sample with identification, date, and experimental conditions. When thawing is required, the specimen is warmed gradually in a 37 °C water bath, monitoring until the core reaches physiological temperature. Rapid thawing minimizes recrystallization, which can damage cellular structures.
Key considerations for successful outcomes:
- Maintain aseptic conditions to avoid contamination.
- Use calibrated temperature sensors for accurate monitoring.
- Verify that the cooling rate matches the intended preservation goal (e.g., high‑resolution histology versus molecular analysis).
- Document all parameters—anesthetic regimen, cooling method, temperature profile, and storage conditions—to ensure reproducibility.