Thomas
I think that you have to be very careful about the perfusion pressure.
If too high then some vessels may rupture. If possible a peristaltic
pump should be used.
The time for flushing out the blood depends largely on what is being
studied and the site for introduction and efflux of the perfusate.
Fluid will tend to bypass some of the vessels especially the smaller
ones if the time of flushing is too short. Look at the area you are
interested in, it should become significantly paler if you have
adequately flushed out the blood. Use heparin and Lidocaine in the
flushing solution.
Barry

Sunil Thomas K wrote:

> Dear Madam,
> Thanks for mailing your opinion. Should try out a
> different PFA powder to see if it gives a better
> result. The fixation is better now. Probably the
> technique.
>
>  How long should one perfuse with saline/PBS before
> switching over to PFA?
>      I switch over after about 5 minutes when the
>     efflux is weakly blood stained (for a 20-30 gm
>       rat pup).
>   How slow should be the flow rate?
>     I guess it should be roughly equal to
> physiological     rate.
>
>   Your thoughts regarding these questions will be most
> useful.
>
> Sincerely,
> Sunil Thomas K
>
> --- Natalie Prigozhina <nataliep@scripps.edu> wrote:
> > Dear Dr. Thomas:
> > Your problem may be in using old PFA powder. I don't
> > think increasing the
> > concentration would help. Try some fresh PFA (at
> > least borrow from somebody
> > a little to check if it gives you better results).
> > Also depending on the
> > structures you are staining, you may be able to use
> > simple methanol
> > fixation (5-10 min in -20C methanol). I am a cell
> > biologist and work with
> > cells cultures. Hope it helps with your tissues.
> >
> > Sincerely,
> > Dr. Natalie Prigozhina
> > The Scripps Research Institute,
> > La Jolla, California
> >
> > P.S. Next time you post in a newsgroup,  consider
> > addressing to people as
> > "Dear Sir or Madam".
> > Good luck with your stainings.
> >
>
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