[Histonet] question about mouse perfusion
jkiernan <@t> uwo.ca
Thu Mar 18 11:45:43 CDT 2010
The volume of a mouse is about 25ml, of which perhaps 2ml is blood. 5ml of saline (or PBS) is enough to wash it all out. Some people precede this with a small bolus (about 0.2ml) of 1% aqueous sodium nitrite to dilate the blood vessels. After washing out the blood, perfuse with about 10 ml of fixative, over the course of 5 to 10 minutes. It's important that the pressure isn't too high, to avoid rupturing capillaries and small veins, and waterlogging (wrecking) the tissues. If perfusing from a syringe, use the thinnest available needle to attenuate the pressure; 10 minutes feels like a lot longer when you're pushing the plunger very slowly and keeping the sharp end of the needle motionless!
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----- Original Message -----
From: Li Zhang <dancingwing <@t> yahoo.com>
Date: Wednesday, March 17, 2010 14:59
Subject: [Histonet] question about mouse perfusion
To: histonet <@t> lists.utsouthwestern.edu
> Dear all,
> I have just started to learn how to do perfusion in mice and
> this site has helped me a lot.
> We don't have the pump or Perfusion one in the lab, and probably
> won't be able to get one in the near future, so I'm using the
> hand injection method. I'm using a 60 ml syringe with 21G
> needle. I am doing perfusion in order to do immunofluorescence
> on the brain.
> My question is: can anyone give me a rough idea of how fast I
> should inject ( like ml/min). I think I've tried like 30 ml in 3
> min, and I suspect that it's too fast because I do observe
> tissue swelling sometimes.
> And another question, which of the following is better for
> perfsusion: 1) PBS and then 4% PFA 2) Saline and then 4%
> PFA 3) sucrose--> saline--> PFA?
> Thank you very much for your help!
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