[Histonet] DIF on paraffin embedded tissue

Wed Nov 25 11:28:12 CST 2015

Also agree. Lots of people do it. Search PubMed, J. Histo Cyto.

If you have the appropriate filters, using Cy dye secondary's can help move away from any autofluorescence.

Brett

Brett M. Connolly, Ph.D.
Prin. Scientist, 
Translational Biomarkers - Imaging
Merck & Co., Inc.
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brett_connolly at merck.com
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-----Original Message-----
From: Marcus Green via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Wednesday, November 25, 2015 12:23 PM
To: Elizabeth Chlipala; Simmons, Christopher; Rene J Buesa
Cc: 'histonet at lists.utsouthwestern.edu' (histonet at lists.utsouthwestern.edu)
Subject: Re: [Histonet] DIF on paraffin embedded tissue

I, agreeing with Liz,

would suggest looking out a IF protocol for FFPE tissues - I've recently concluded a validation of PLA (proximity ligation assay) in: cells in culture, FFPE cell pellets and FFPE xenografts. The signal was consistently there (although a drop in expression - from fixative reasons as well as culture conditions). Background is the biggest issue for IF using FFPE so make sure to run some comprehensive controls.

You will need to run a Deparafinisation, Dehydration and Rehydration as you would IHC (3mins xylene x2, 3mins 100% EtOH, 3mins 70%EtOH and 3mins 50%EtOH - or similar) before running an Antigen Retrieval - if you have this optimised for IHC use something similar (pH and time). I would also consider a permeabilization step and if you need to optimise for background (Sudan black wash).

If, however the question is can you reinstate a tissue back to an unfixed tissue for freeze processing then no you can't.

hope this helps,
kind regards,

Marcus

________________________________________
From: Elizabeth Chlipala via Histonet [histonet at lists.utsouthwestern.edu]
Sent: 25 November 2015 16:53
To: Simmons, Christopher; Rene J Buesa
Cc: 'histonet at lists.utsouthwestern.edu' (histonet at lists.utsouthwestern.edu)
Subject: Re: [Histonet] DIF on paraffin embedded tissue

I'm not sure that is the case in the grand scheme of things, it will depend upon the target that you need to stain via immunofluorescence.  Technically we perform IF staining on frozen sections primarily because the antigen does not survive formalin fixation.  Many people utilize IF techniques on FFPE tissue with good success, we have done it here.  There are things you will need to consider and what I suggest below may not work at all.  It's up to you if you want to try it or not and you may feel it is not worth the time and energy required to see if it may work on FFPE samples, it could be a lot of work and it may not be successful.

1.  You will likely not be able to use your current protocol
2.  You will likely need to add an antigen retrieval step
3.  You may need to look for a different antibody source (one that survives formalin fixation)
4.  The signal needs to be good since formalin fixation will cause autofluorescence

Unless I am completely missing something here since two individuals have stated that the sample is useless, maybe there is a better explanation as to why the sample is completely useless.  Here is where I am coming from - technically you can perform IF staining on FFPE tissue samples, people do it all of the time, we have done it here, it's a common technique for dual labeling of samples when co-localization is an expected result.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz at premierlab.com
www.premierlab.com

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-----Original Message-----
From: Simmons, Christopher via Histonet [mailto:histonet at lists.utsouthwestern.edu]
Sent: Wednesday, November 25, 2015 9:10 AM
To: Rene J Buesa
Cc: histonet at lists.utsouthwestern.edu
Subject: Re: [Histonet] DIF on paraffin embedded tissue

Useless sample
Sorry

Sent from my iPhone

> On Nov 25, 2015, at 11:02 AM, Rene J Buesa via Histonet <histonet at lists.utsouthwestern.edu> wrote:
>
> No matter WHO to tell you to do WHAT, for IF purposes, that FFPE
> tissue is USELESS.René
>
>
>    On Wednesday, November 25, 2015 10:55 AM, Maryann Deathridge via Histonet <histonet at lists.utsouthwestern.edu> wrote:
>
>
> We have a tissue sample that was processed and paraffin embedded.  We
> URGENTLY need to recover the tissue and perform Immunofluorescence on
> the sample.
> Does anyone have a procedure.  HELP
>
>     madeathridge at pastnashville.com
>
>
>
>
>
>
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