Successful IHC with Re: [Histonet] paraffin vs. frozen

Gayle Callis gcallis <@t>
Tue Mar 6 14:40:01 CST 2007


What species are you working with?  And what markers?  Basically, it comes 
down to fixation of the antigen - whether IHC will be successful after 
crosslinking fixatives followed by antigen recovery (not always needed) or 
having to use another type of fixative to reduce aldehyde crosslinking 
(Paraformaldehyde-llysine- periodate (PLP), or avoid cross linking entirely 
and resort to precipitating fixatives (cold acetone, acetone/absolute 
ethanol for murine CD markers) or a Zinc TRIS Buffer formalin free fixative 
developed by Beckstead.  Unfortunately, one thing for all doesn't work for 
some of the touchy antigens.

If you are working with murine tissue, and doing CD markers, it is very 
common to have many antigens (CD markers) totally compromised by any 
aldehyde fixation with either formalin or paraformaldehyde followed by 
paraffin processing.  The worse scenario is needing several markers on one 
experimental tissue and two markers will not stain after formalin fixed 
paraffin embedded tissues (FFPE) even after retrieval or digestion 
methods.   This is the reason we do cryotomy for 99.5% of our murine work.

There are going to be times when FFPEwill not work with other species 
either and you have to use cryotomy.  Due to this, we simply gave up using 
FFPE, and now exclusively do fresh tissue frozen sections.   The nice thing 
about the latter, if one like flourescent work, there is no aldehyde 
induced autofluorescence to deal with.

Good luck

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

  At 11:22 AM 3/6/2007, you wrote:
>If anyone could give some insight as to what the differences are between
>frozen sections and paraffin sections when performing IHC I would
>greatly appreciate it.  I am running a study and the results for a few
>of the markers I am not to happy with and am contemplating changing over
>to frozen sections for upcoming studies.  Is there any rhyme or reason
>as to why antibodies work better with frozens sections or paraffin
>sections?  Any help will of course be greatly appreciated.
>Kris Kalleberg
>Research Scientist
>Unilever R&D
>40 Merritt Blvd.
>Trumbull, CT 06611
>(203) 381-5765
>Histonet mailing list
>Histonet <@t>

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