[Histonet] Fixing question

[Rene J Buesa]

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 <cfranci <@t> rigel.com> 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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