RE: [Histonet] Microwave Processing

From:"Barry R Rittman"

The term fixation is a generic one that is a greatly misused one in the literature.
We use the term fixation to include the following three steps.
1.	When a fixative reaches tissue there is an in initial "killing" of the cells.
2.	This is followed by a "stabilization" of components. The bonds that are formed are often temporary bonds that can be broken. (An example is the use of formalin for a short time of 1-2 hours, wehen the "temporary" bonds  that have been formed can usually be broken by washing in running tap water.). This has been used to advantage in histochemistry when enzymes are prevented from diffusing but can still be demonstrated.
3.	If tissue is left for a sufficient length of time then "fixation" occurs. The bonds that are formed are more permanent, more stable and often require the use of procedures such as antigen retrieval to break these bonds. 
This last step continues sometimes for years with more and more permanent bonds formed. This results in the increasing difficulty of staining tissues that have been fixed for a long period of time.
It is important therefore when discussing fixation to note the duration of "fixation" and the specific conditions under which the fixative is applied.
Barry


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Marshall Terry Dr,Consultant Histopathologist
Sent: Wednesday, April 21, 2004 6:48 AM
To: Steven E. Slap; kevin williams; histonet@pathology.swmed.edu
Subject: RE: [Histonet] Microwave Processing


My confusion has not been dispelled.

Steven writes:

"The saline will not work 
alone as a fixation step, except in the case of 
very small biopsies (as written up in an article 
by Tony Leong)- ..." 

In the next post, we get:

"Yes, in the Leong method for biopsies, the specimens are heat 
stabilized in the microwave in saline, and not really chemically 
fixed.  They get fixed in the ethanol in a traditional processor ..."

These statements are mutually incompatible.

Moreover, what is the difference between "stabilisation" and "fixation".

A further muddle is introduced by:

"not really chemically fixed."

Well, hell, are they fixed or no? Heat fixation is fixation - who cares whether chemically fixed?

Of course, the major problem is that mechanisms of fixation are varied (with the fixative) and not well understood. (Speak for yourself do I hear someone say?)

Dr Terry L Marshall, B.A.(Law), M.B.,Ch.B.,F.R.C.Path  Consultant Pathologist  Rotherham General Hospital  South Yorkshire  England
        terry.marshall@rothgen.nhs.uk

-----Original Message-----
From: Steven E. Slap [mailto:siksik03@comcast.net]
Sent: 17 April 2004 22:54
To: Marshall Terry Dr, Consultant Histopathologist; kevin williams; histonet@pathology.swmed.edu
Subject: RE: [Histonet] Microwave Processing


Hi Terry & HistoNetters

I have done lots of successful large organ 
stabilization in saline at 65C for 20-35 minutes 
(depending on the size of the organ.  However, it 
is important to emphasize that the purpose of 
this step usually is to firm up the tissue so 
that it can be subsequently cut up into thinner 
pieces for processing, which must include a 
fixation step, either in the microwave or on a 
conventional processor, in formalin or some 
non-formalin fixative.  The saline will not work 
alone as a fixation step, except in the case of 
very small biopsies (as written up in an article 
by Tony Leong)-  a procedure I have not tried 
myself.

best regards,
Steven Slap
Microwave Consultant


At 3:58 PM +0100 4/16/04, Marshall Terry Dr,
	Consultant Histopathologist wrote:
>Sorry to but in - but on a related question -
>does anyone do microwave fixation in saline as 
>described by for instance the Melbourne group, 
>which involves bringing up to 65C in saline?
>Have tried it this week and the sections are even crummier than usual.
>When I was in Tasmania, a local private lab. did 
>it and their sections were fine.


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