Hi Patsy,
Like Luis already answered, double staining is the best way to identify 
caspase-3 positive cells. I have done this recently and indeed it 
worked very well. You are lucky in the first place that caspase-3 is a 
rabbit antibody that is easily combined with any mouse primary. Whether 
or not the cellular make-up during apoptosis is changed you never know 
before. This is just a matter of testing. I can assure you from my own 
experiments that double staining of caspase-3 combined with CD31 or SMA 
works for sure. Since caspase-3 shows both nuclear and cytoplasmic 
staining I stained CD31 or SMA with beta-galactosidase in turquoise and 
caspase-3 with alk. phosphatase in red (DakoCyto Liquid Permanent Red). 
If caspase shows up in the cytoplasm of a b-gal positive cell, co-
localization is observed (blue/purple mixed-color). Optional is a very 
faint hematoxilin counterstain (use 1/10 diluted hematoxilin only for 
15 sec or so) or Nuclear Red (15 sec). A very colorful experiment I can 
tell you!
Hope this helps.
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center
Amsterdam - The Netherlands   

----- Original Message ----- 
>From  Patsy Ruegg  
Date  Mon, 22 Nov 2004 11:40:54 -0700 
To  'Histonet'  
Cc  IHCRG@neo.agsci.colostate.edu, jenrueyt@hotmail.com 
Subject  [Histonet] how to identify what cell type is caspase3+ 

I have a question from an investigator who is looking at rat lungs for
apoptosis by using cleaved caspase 3 IHC and then wants to know what
cell type is under going apoptosis (are they endothelial cells or a mix
of different cells? how can they demonstrate the cell type that is
positive for CC3 since the morphology changes when this happens)  they
do not appear to be smooth muscle cells since there is no positivity
around the arteries.  Should be be doing double IHC labeling say CC3 and
CD31 or F8 together in the same section?  Will the cell undergoing
apoptosis still be expressing these other markers?
Patsy

----- Original Message ----- 
>From  Luis Chiriboga  
Date  Mon, 22 Nov 2004 14:08:20 -0500 
To  Patsy Ruegg , 'Histonet' 
 
Cc  IHCRG@neo.agsci.colostate.edu, jenrueyt@hotmail.com 
Subject  [Histonet] RE: [IHCRG] how to identify what cell type is 
caspase3+ 

Hi Patsy
Yes, That's exactly the approach we use.....double labeling should 
catch all
CC3/Surface Marker+ cells except for those that have lost significant
fractions of their cell membrane.
Luis







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