It is important to understand and control the pressure at which you are
perfusing.  A syringe leaves you in doubt, gravity flow is frequently
not enough pressure.  

An EM microscopist named Cragg developed the pressure procedure
mentioned at the following link, and I developed the product offered for
consistent and shrink free perfusions.

http://www.myneurolab.com/mnl/mnlsite/ViewProduct.asp?idproduct=471001&c
atdesc=Histology+Equipment&subcatdesc=Sacrifice+Equipment&idsubcategory=
21


Cordially,

Charles W.  Scouten, Ph.D.
myNeuroLab.com
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300  
FAX  314 522 0377
cwscouten@myneurolab.com
www.myneurolab.com


-----Original Message-----
From: Monson, Frederick C. [mailto:fmonson@wcupa.edu] 
Sent: Thursday, February 06, 2003 7:27 PM
To: 'Martin, Ronald'
Cc: List-HistoPath (E-mail)
Subject: RE: mouse perfusion

Hi Martin,
	I have perfused mice, rats and rabbits, used different routes,
but I
have NEVER gone to the trouble of preparing HCHO from paraformaldehyde
and
then transferred the tissue to Formalin.  I can only say that it in my
experience, I have transferred to the same fluid with which I perfused,
at
least for 1-2hr with small pieces, unless I was performing a double
fixation.  
	If one treats the processes of histologic technique as parts of
the
experimental routine, then it makes little sense to introduce another
variable by using more fixative formulations than are absolutely
necessary.
	Finally, the purpose of perfusion is to perform rapid, holistic
fixation on a target of interest.  While the kidney is a good organ to
use
as a test of aortic perfusion, if the initial purge of blood is not done
quickly with sufficient pressure the kidney will remain filled with
clotted
blood.  The liver would be better fixed by perfusion via the portal vein
rather than the aorta.  
	Kidney and liver cannot be fixed well by immersion after
perfusion
if the perfusion did not work, and even for immersion fixation alone,
the
organs should not be fixed whole - even from a mouse.
	I would recommend the text on electron microscopy by Hayat as a
good
reference for good perfusion methodology.  Electron microscopists have
evolved very useful methods for fixing specific tissues by perfusion.
	As gently as possible, I would recommend to your P.I., a good
read
of the literature and/or the book mentioned above.
	Ah, anatomy.  There was a time when everyone who worked in this
business took at least one course in anatomy.  Now, far too many back
into
the subject and never do get themselves tuned in to see how the
structure
and function of the 'cellular' organism is related to the structure of
its
larger parts.
	I can only conclude that the kidney and liver were not perfused
properly and then couldn't be fixed any better in the subsequent
formalin
'bath'.

Hope this helps,

Fred Monson

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging

Mail to:
Geology, CASI
West Chester University of Pennsylvania
Schmucker II Science Center, Room SS024
South Church Street and Rosedale Avenue
West Chester, PA, 19383

Phone & FAX:  610-738-0437
eMail:  fmonson@wcupa.edu

For help and information only,
The CASI houses:
An FEI Quanta 400 and Technai 12T, 
Oxford INCA Energy 400, 
Tousimis AutoSamdri 815 and 
Olympus FV-300.
  


-----Original Message-----
From: Martin, Ronald [mailto:Ronald.Martin@umassmed.edu]
Sent: Thursday, February 06, 2003 1:39 PM
To: histonet@pathology.swmed.edu
Subject: mouse perfusion


Fellow Histonetters,
I have a question regarding tissue fixation via perfusion. I have a PI
who
has, on two occcasions, submitted mouse tissue that has been perfused
(liver, kidney, skin, small bowel-whole body perfusion). I have found on
both occasions that the tissue (particularly the liver and kidney) seems
very dry. I have processed other tissue for her that has not been
perfused
but fixed in 10% NBF  (Fisherbrand) with very good results using the
same
tissue processor and processing schedule.
My question is -could this perfusion technique be over fixing the
tissue? I
think she uses formalin (lab made from paraformaldehyde) for the
perfusion
then continues to fix the tissue in formalin (Fisherbrand) for 4 hours
after
harvesting it. Do I need to compensate for this procedure by shortening
my
tissue processing schedule? 
I have looked through the histonet archives and found some information.
Any
suggestions are greatly appreciated.
Thanks in advance,
Ron Martin, HTL (ASCP)HT
Research Associate