[Histonet] CD4 CD8 in FFPE murine tissue

[Gayle Callis]

Dear Jackie,

There are no miracles for FFPE mouse tissues and CD4/CD8 including a 
miracle antigen retrieval or enzyme digestion that will recover these 
antigens after formalin or paraformaldehyde  (the enemies)!  This has been 
discussed innumerable times on Histonet, and so far, NO ONE has come up 
with a solution for FFPE CD4 and CD8 success.  It is an unfortunate fact of 
murine IHC life.

You have  options.

If you insist on paraffin, you can only use the zinc tris buffer fixative 
(formalin free) which you can make up yourself.  Tissues have fairly good 
morphology, but not equivalent to NBF fixed tissues.  However, you can do 
CD4 and CD8, but it is advisable to use alk phos enzyme IHC since endog 
peroxidases are horrible, cannot be quenched after this fixation.   The 
tissues are drier, so careful limited processing is a good idea, and lots 
of block face soaking before sectioning. BD Pharmingen sells the fixative, 
but it is really easy to make up in house. I will be happy to send method 
privately.  Antibody concentrations are fairly close to frozen section 
work.  I was not impressed with sectioning after this fixative and thought 
the tissue looked dry, so all the publications on this that tout equivalent 
morphology as NBF have not proved to be entirely accurate.

I don't know if PLP fixation would help either, I have seen little success 
with this fixative.

The best and other alternative is to do fresh snap frozen tissues.  This is 
all we do with all our murine tissues, since we have to do CD4 and 8 along 
with several other CD markers on a single sample of tissue.  This totally 
rules out FFPE for what we do so we don't even bother with NBF.  Our 
morphology is excellent along with wonderful IHC staining using 75% 
acetone/25% absolute ethanol (A/A) fixation.  You cannot substitute alcohol 
and you cannot do harsh endog perox block - use DAKO S2001 peroxidase block 
instead to save the section from bubbling off a slide.  Some people use 
acetone, but we have found AA to be superior.  If we do acetone, it is a 
double acetone fixation. Air dry overnight, 4C acetone 10 min, air dry 15 
min in front of fan, return to 4C acetone for 10 min, air dry, rinse off 
OCT and proceed with staining.   If you are doing large studies, there are 
some snap freezing methods that allow you to handle large load freezing 
sessions and not use liq nitrogen cooled isopentane or hexane.

Method: snap freeze tissues, air dry FS overnight at RT then fix in 
Acetone/alcohol fixative for 5 min at RT, go directly to buffer rinse X 
2.  DO NOT redry sections after this fixation.  Proceed with staining 
includine DAKO perox block, and avidin/biotin blocking.

I have one method using A/A fixation, using biotinylated CD4, then 
Strepavidin-HRP, DAB+ from DAKO and DAB enhancer from DAKO.  I had to use 
the glucose oxidase endogenous peroxidase block, but I had my primary 
antibody diluted out 1:15,000 and fading at 1:20,000.  This is something 
you never see with FFPE, and probably not with ZnTris buffer fixative either.

I suggest you use the Permanent Red (new from DAKO) as it is more sensitive 
(Chris van der Loos would say efficient) with the ZNTRIS fix, you probably 
will have even lower primary concentrations.

Good luck from one of Santa's murine IHC elves



At 09:25 AM 12/22/2004, you wrote:
>Does anyone have any Christmas miracles for CD4 and CD8 in FFPE mouse?
>
>Jacqueline M. O'Connor HT(ASCP)
>Abbott Laboratories
>Global Pharmaceutical Research and Development
>Discovery Chemotherapeutics
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