[Histonet] RE: Dead-End system & BMA/MMA embedded tissue

[C.M. van der Loos]

Dear Sven,
I can just confirm your observation:
Once I tried to do a TUNEL staining on methacarn-fixed, paraffin-embedded(=similar to Carnoy, except the alcohol is here replaced by mehtanol) tissue along with standard formalin-fixed tonsil as positive control. The apoptotic nuclei in the formalin-fixed tissue were nicely stained but the staining of the methacarn-fixed tissue was terrible as you described. Apparently, the TUNEL staining is not fitting with this alcohol-based fixative.
Hope this helps,
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center
Amsterdam - The Netherlands    

----- Original Message ----- 
>From  Sven van Eijl <T.J.A.vanEijl <@t> pharm.uu.nl> 
Date  Fri, 21 May 2004 17:28:39 +0100 
To  histonet <@t> lists.utsouthwestern.edu 
Subject  [Histonet] Dead-End system & BMA/MMA embedded tissue 
Dear Histonetters,

This will be my first posting to this list, and I am by no means a real histologists, so bear with me please...
We are having a problem using Promega's Dead-End colorimetric TUNEL kit on plastic embedded lungs.
In the manual are only guidelines for formalin fixed and parrafin embedded or frozen tissue. We fixed the lungs using Carnoy's and embedded in BMA/MMA. I have had some tips from Promega about modifications to the protocol to account for the use of other fixatives than formalin, but am wondering if it is the fixative or the plastic that is causing the problems. We get a nice brown staining of all nuclei at all times, also in the negative controls. It is not a question of general high background, the rest of the tissue is clear. Any suggestions are highly appreciated.

Thanks,
Sven