Blood serum samples are collected at P60, P90 and P140

Blood serum extraction
Butterfly catheter
Heparin 1:10 solution
Microtainer SST
Heated cushion
Rat immobilizer
Masking tape
Water
Electrical water heater
Alcohol and cotton

Before starting the procedure, remember that when changing the housing room, the rats need 1 hour to get used to the new location (it is important to take this into account if you are interested in controlling the stress and novelty factor).

1. A 1:10 heparin solution is prepared to heparinize the butterfly catheters.
2. The immobilized rat is placed on the heated cushion to facilitate vasodilatation.
3. Heat water to 36ºC if necessary to facilitate vasodilation. Place it in a container that allows the rat's tail to be inserted.
4. Place the rat in the restraint and secure it.
5. Take the distal third of the rat's tail and make sure to identify the vessels, if you are able to do so with the naked eye it means that adequate vasodilation has been achieved.
6. The veins are those found on the sides of the tail, avoid taking your sample from the artery, it is the one that is centered on the dorsum of the tail.
7. Take the tail between the thumb and middle finger of your non-dominant hand, and with the index finger present the tail in the direction of your location.
8. With your dominant hand take the tube with the catheter needle and with the bevel upwards, puncture the vein at an angle of <45º.

Remember that you can rearrange the rat or its tail as many times as necessary so that you have visibility of the procedure. The rat will be fine as long as the procedure is done properly.

9. The veins are superficial so it is not necessary to puncture very deep. When you have managed to find the vein, a small drop of blood will be seen through the base of the catheter.
10. Refill as many times as necessary, without removing the needle. It is recommended only two puncture attempts are to avoid further discomfort.
11. 400 mL is sufficient for analysis.

Figure from Hatakeyama, S., Yamamoto, H., & Ohyama, C. (2010), it is not necessary to modify the needle as seen in the figure.

Configuration of a butterfly catheter fitted to a microtainer SST tube that with the help of masking tape or micropore tape is kept together adapted by BSc Diego Ortuzar-Martinez

Ethanol kit assay

References

Hatakeyama, S., Yamamoto, H., & Ohyama, C. (2010). Tumor formation assays. In Methods in enzymology (Vol. 479, pp. 397-411). Academic Press. https://doi.org/10.1016/S0076-6879(10)79023-6