ER/PR on archive material

[philtrougouboff]

Have someone experience or knowledge  :idea:  about the stability of ER/PR receptors in paraffin blocks (Breast Carcinoma)? I do a new stain with the same Ab on block  that was strongly ER & PR positive in 1998 and now the new immunostain is nearly negative :!:  :?: The 1998 immunostaining was "manual" and the 2005 stain was made with an automatized stainer (Ventana's Benchmark) with a positive control that was ok. A p53 stain (2005) of the same block was strongly positive! Perhaps do you have a reference about this problem?
Thanks for any help. Phil

[ole]

Hi Philippe
 I have not seen that loss of reactivity of ER and PR is reported in FFPET archieve material. I have done ER/PR on several old paraffin blocks, and i find the signal often is stronger (/ue to improved technique and antibodies)

  I have seen (reported) some clones of AR are affected by this, but not ER and PR.
But (at least some clones ER/PR) on mounted sections the signal/antigenisity is getting weaker from about a weak stored in RT. On stored (old) paraffin blocks i trow away the 3-4... first sections.
Perhaps if the tissue is poor fixed and processed the stability of the ER/PR receptors gets affected :?:  i dont know.

Interresting question. Please reply if you find some info about the subject.

Ole

[philtrougouboff]

Thanks a lot for your answer. I still haven't found what I'm looking for (quoting U2) on this subject. Perhaps the problem comes from poor conditions of storage...It's very hot here more than half of the year.
Perhaps as you wrote, use of different technic or clone :?:
To be continued I hope  

[ole]

Hi
Yep some antibodies work better on "poor" processed/autolytic material. i did a lot of immuno on such material some years ago. I found some polyclonal Ki67 to be better than the monoclonals we had. It seems to me that eg ER (clone sp1) from neomarkers are a bit better on these kind of tissues than some of our ER mouse monoclonals.

Ole

[philtrougouboff]

Thanks for the clone's reference
Phil

[madeleine huey]

Hi,
ER/PR are not recommend baking too long (bake no longer than 1 hr @ 60C).  Here's my experience for these 2 markers.  1) Bake paraffin section only same day you use  2) Never bake more than once (some laboratory still practice baking for than once after cutting, and leaveing sections in RT for months & years).  
You might want to re-cut some fresh section & try it with the old ab.  I beg you will get the stain this time.
Let me know if they don't work.
Madeleine

[ole]

Hi Phil
im currently reading thru my old articles, found an article that "touches" the issue.
Even though the article is about reactivity of stored paraffin slides, they compare with recut slides (old blocks), .... some interesting thoughts about storage etc. Perhaps you could store a few blocks cold a few years and compare with some stored "hot" (your warm climate).

in case you have not read it........

Bertheau P, ........
Variability of immunohistochemical reactivity on stored paraffin slides.
J Clin Pathol 1998; 51: 370-374.

I have a good article somewhere about "what affects ER activity", it doesnt mention storage PET as an factor,  but ill find it.

Ole

[ole]

Phil
Was not quite what thought i remembered "the article"  :oops: but still........

Daniel A Arber.
Effect of Prolonged Formalin fixation on the immunohistochemical Reactivity of Breast Markers
Applied immunohistochemical and mol morp 10(2): 183-186, 2002

If you have tried it?
I find the SP1 antibody to work best with AR (superheating in TE pH 9), even though both articles about the "novell" antibodies and perhaps datasheet claims that it works optimal also without AR.

Ole

[philtrougouboff]

Thanks a lot for the references.I'll have a look soon.
Phil