RE: [Histonet] RE: caspase 3 and pcna

From:Margaret Blount

Dear Chris,

I read your message with interest as I am setting up a new histology lab and
anticipate that I will need to be able to do these techniques on rodent
tissues. I have been working with human skin for a long time and need to
refresh my memory on animal samples.

You mention HIER on FFPE sections for Caspase-3, but do not state how long
you perform this step. Do you use a microwave or pressure cooker? Is it a
short cycle or longer? 

Thankyou for your valuable contributions to histonet.


Margaret Blount
Chief Technician
Clinical Biochemistry
University of Cambridge
Addenbrooke's Hospital
Hills Road
CB2 2QR 

-----Original Message-----
[]On Behalf Of C.M. van
der Loos
Sent: Tuesday, June 01, 2004 3:17 PM
Subject: [Histonet] RE: caspase 3 and pcna

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' 
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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