Re: [Histonet] Fixing question

From:John Kiernan

A little clarification is called for here.

1. The diffusion of a fixative into a specimen follows the
law of diffusion:
                   d = K sqrt(t)
where d is the distance penetrated (mm) and t is time (hours).
[sqrt(t) means the square root of the time.]
For formaldehyde entering a gelatin-albumin gel the value of 
the constant K is given by JR Baker as 3.6 (Principles of
Biol. Microtechnique, p.40). 

2. Although
 formaldehyde penetrates rapidly its chemical
reactions with proteins occur rather slowly. Studies with
radioactively labelled formaldehyde indicate that the
amount bound reaches a maximum at 24 hours. Most of this
can be removed by prolonged washing in water and probably
is not involved in cross-links. Cross linking, which 
increases the resilience of the tissue in the face of
osmotic gradients, alcohols etc, takes about 2 weeks,
but is often considered to be sufficient after 24 hours.
With shorter times in formaldehyde, tissues are still
osmotically responsive, and subsequent immersion
alcohol causes coagulation of proteins that have not
been cross-linked. This is not necessarily a bad thing.
Cell nuclei stain more strongly after coagulant fixation
than after thorough fixation with formadehyde.

For a short account of how aldehyde fixatives work, see
For more details, Pearse's Histochemistry 4th ed Vol 1, 
or Hopwood's chapter in the 2002 edition of Bancroft &
Gamble's Theory and Practice of hisatol. Techniques. 

John Kiernan
Anatomy, UWO
London, Canada

Rene J Buesa wrote:
> Christian:
> There are several issues to consider including the type of fixative. I you are referring
> to neutral buffered formalin (NBF) overfixation occurs when tissues are left more time
> than necessary and this will depend on the thickness of the tissue slice to be fixed.
> NBF has a diffusability coefficient (K) of 0.78 mm/hour so a slice 4 mm thick will require
> 2.5 hours to fix (remember that the NBF will be "entering" the tissues from both sides!).
> Any time after that will be "theoretically" and overfixation, i.e. not needed time in NBF
> and this can translate into more crosslinkage of the proteins and more difficulties for
> immunohistochemistry (IHC) procedures.
> Ethanol has a K of 1.0 mm/h so the same type of slice will require only 2 hours, will
> fix by quagulation and theoretically will not interfere with IHC but, again, after 2 hours
> it is not necessary to keep the tissue in ethanol.
> Having said all that, in tissue processors like the Sakura VIP all stations can have
> vacuum/pressure only depending in how long the station is programmed to last.
> With thin enough slices of tissue I don't think you have to worry about overfixation
> as long as you keep the tissues in the fixative the time required for its penetration,
> whichever the fixative is.
> Hope this will help (although I think I gave a somewhat "convoluted" answer!).
> Rene J.
> Christian Franci  wrote:
> dear folks, pardon my ignorance but....
> It seems that pressure is applied to the processing of tissues to
> ensure evenness and to force out any residual alcohol and/or xylene
> that might over-dry one's delicate samples.
> I gather that depending on what machine you have you can apply pressure
> at each step or just at the final paraffin stage ( as in my case).
> Here is my question... would applying pressure during the fixation of
> the tissues also be beneficial?
> Would it speed up the process therefore minimizing the possibility of
> over-fixation?
> Do any of you fix tissue under pressure and, if so, how do you go about
> it? Just curious....
> Cheers
> Chris
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