[Histonet] question about mouse perfusion

[Merced M Leiker]

Hi Li,

Try 1-2 ml/min perfusion rate. Yes 10ml/min is way too fast. I know it's 
tricky; I've done it by hand and by pump.

Perfuse with PBS or Saline (same thing) first, then 4% PFA til mouse is 
stiff, then after taking the brain out immerse it in  increasing grades of 
sucrose (10%-->20%-->30%) at 4C til brain sinks to the bottom of the vessel 
each time. Takes longer to sink with increasing grades - I think if I 
remember correctly in 10% it's only half an hour, 20% is a few hours, and 
30% is overnight. Something like that. Some people skip the 20% and just do 
10% and 30%. Some people just do 30%. Sucrose helps cryopreserve the 
delicate brain tissue.

Regards,
Merced

--On Wednesday, March 17, 2010 11:58 AM -0700 Li Zhang 
<dancingwing <@t> yahoo.com> wrote:

> 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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Merced M Leiker
Research Technician III
Cardiovascular Medicine
348 Biomedical Research Building
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214  USA
leiker <@t> buffalo.edu
716-829-6118 (Ph)
716-829-2665 (Fx)

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