[Histonet] RE: caspase 3 and pcna

From:"C.M. van der Loos"

Dear Patsy,
We used a anti-caspase-3 rabbit antibody from Cell Signaling (#9661) on FFPE sections in a 1/200 dilution (60 min, RT) after HIER with Tris/EDTA pH9.0. This means that the epitope survives both formalin-fixation and embedding through alcohols. In your situation you may to fix the cells at the chamber slide with 4% PFA (or routinely buffered formalin) for 5 min and perform your caspase-3 staining. As it is nuclear staining perhaps you need to add 0.1% saponin to all antibody steps and washing buffers to open up the cell membranes letting your antibodies go in and out.
With respect to PCNA (we call it a poorman Ki67!!) you may try to replace it by anti-Ki67, rabbit monoclonal SP-6 (Neomarkers). In our hands it reacts with human, mouse and rat proliferating nuclei on FFPE's and cryo's without any non-specific background staining.
Hope this helps!
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center
Amsterdam - The Netherlands   

---- Original Message ----- 
>From  Patsy Ruegg  
Date  Thu, 27 May 2004 13:09:18 -0600 
To  Jackie.O'Connor@abbott.com 
Cc  histonet@lists.utsouthwestern.edu 
Subject  [Histonet] caspase 3 and pcna 
Jackie and all, could you please advise me on doing caspase 3 and pcna on
cells grown on chamber slides, I mostly need advise with caspase, what
antibody do you use?
Thank you all,

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