[Histonet] question about mouse perfusion

[John Kiernan]

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!
 
John Kiernan
Anatomy, UWO
London, Canada
= = =
----- 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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