I am trying to remove the blood from the brain capillaries after injection of a dye, I tried intracardial injection of Ice-cold heparinized PBS but I faced high resistance during the injection, so if anyone can help with a detailed protocol to make sure that whole blood was removed from the brain capillaries this will help a lot.
Your suggestions are much appreciated.
Thanks
Dear Sweilem,
Intracardiac perfusion is the best way to remove the blood from brain capillaries. You will need to add sodium nitrate and heparin to your PBS solutions to prevent clothing. You will need to use a large needle (blunt, >G18) to reduce the resistance you have mentioned. Basically deeply anesthetise the animal, open the chest cavity, expose the heart and stabilize it with forceps, insert your blunt needle up from the ventricle, start the flow of heprinized PBS solution with a pressure at around 150-200 mmHg, cut the right atrium to let the incoming blood out, run the solution until the outflow is clear of red blood cells (~250 ml). If you need to fix the tissue you can change from PBS to 4%PFA (or whatever fixative you are using). For details of the protocol please see my PhD thesis and JCN publication listed below.
best wishes, Refik
Thesis Molecular physiology of ATP-gated ion channels assembled fro...
Article Distribution of the P2X2 receptor subunit of the ATP-gated i...
Hello Sweilem
I totally agree with Refik.
We use a mice feeding needle for this purpose and a perfusion pump (Watson-Marlowe). We perfuse the rats for 5 minutes with PBS before switching to PFA.
Greetings
Dear Dr.Rafik
Thank you very much for your detailed and very helpful reply, and yes I will follow the same protocol you suggested.
Much appreciated,
Sweilem
Thanks Dr.Gasis,
Yes you are right I will follow the same protocol.
Thanks a lot
Sweilem
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