The most important points in the perfusion are to flush out the blood
carefully with saline before perfusing with the NBF, use a muscle
relaxant such as lidocaine, use an anticoagulant such as heparin,
perfuse until fluid is obviously formalin. can always use a weak dye
such as methylene blue to determine the end point. A peristaltic pump is
also highly recommended to prevent excessive pressure.
As the perfusion is only an initial partial fixation, the time after
perfusion and before preparing blocks is the critical time.
Barry

V#233#ronique Wunderle wrote:

> Dear all,
> we are intracardially perfusing our mice with 4%PFA
> before dissecting out organs such as brain and bone
> marrow.
> So here is my questions :
> do you use a standard volume of PFA (ex 30ml) to get
> reproducible conditions ?
> I was just wondering if the volume of PFA perfused
> would infuence the subsequent time of post-fixation of
> the tissues after dissection. As I want to do
> beta-galactosidase staining on frozen sections, I am
> worried about over-fixation and decrease of staining.
> Can anybody give me some exemples of fixation
> conditions of various tissues prior
> immuno-histochemistry or B-gal staining on frozen
> sections ?
> thanks in advance for your help.
> veronique.
>
> =====
> Veronique M. Wunderle, PhD
> Responsable du reseau Nucleis
> tel (0)2 41 35 56 82
> fax (0)2 41 35 41 38
>
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