This is a reply to Christine Kleins message of 9/24/01 concerning Caspase 3. 

She suggested using R&D activated caspase 3 catalogue # AF835.  Steam Ag 
retreival for 25 minutes, primary Ab incubation of 1 hour, Dako LSAB+ kit for 
for secondary and SA-HRP for 15 minutes.  Dab for 15 minutes. 

I used this caspase 3 from R&D and followed your protocol except I used Dako 
EnVision+ for detection and also included DAKO's protein block for 10 minutes.  
I don't know if this makes a difference?  I also was wondering what your 
dilution of caspase 3 was?  I am using it at 1:750.  I am also getting a lot of 
background with positivity so I don't know if I need to increase my dilution 
farther or if I need to increase rinses between steps?  Is it possible to rinse 
to much and wash away your primary antibody and secondary antibodies when 
trying to clean up background?  Thanks again for your help. 

Bob Meyer, HTL 
Northwestern University 

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