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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