Antibody-Based Technologies => Tumor Markers Review => Topic started by: coosbay25 on April 24, 2004, 03:00:50 AM

Title: Anti-caspase 3 for apoptosis in brain
Post by: coosbay25 on April 24, 2004, 03:00:50 AM
Just wondering if anyone has any advice on a good antibody to cleaved caspase 3. We want to use it as a marker of apoptosis, but are getting quite widespread reactivity even in control brain. Anyone with some experience using antibodies to caspase 3 in brain would be great!
Title: Re: Anti-caspase 3 for apoptosis in brain
Post by: richard03 on September 27, 2007, 07:41:48 PM
This one has worked for rat brain nicely.

http://www.ihcworld.com/_protocols/antibody_protocols/caspase3_r&d_systems.htm
Title: Re: Anti-caspase 3 for apoptosis in brain
Post by: dutchmaninvienna on October 09, 2007, 07:01:01 AM
but are getting quite widespread reactivity even in control brain.

This is a major issue in apoptosis research. There are plenty of scientific papers which show such kind of staining in otherwise normal nuclei and call these cells apoptotic cells. I think the staining of normal nuclei is real and not background or an artefact, however, real apoptotic cells in the CNS show caspase-3 in the cytoplasm. Most of the time the nuclei of these cells reveal condensation, but not always ( depending on the form of apoptosis). We have always used the CM1 antibody which we got non-commercially from IDUN. For a certain time this antibody was sold by BD Pharmingen, but not anymore. BD Pharmingen however sells other activated caspase-3 antibodies which in our hands give the same results. I think however that your antibody is working perfectly. We occasionally  also see such (formalin-dependent) nuclear staining with our caspase-3 antibodies (especially in rat and mice). If this nuclear staining is very strong you might dilute your antibody some more. Real apoptotic cells then will still be strongly positive. You can check out my recent for examples of apoptotic cells in the brain:

Bauer J, Elger CE, Hans VH, Schramm J, Urbach H, Lassmann H, Bien CG. Astrocytes are a specific immunological target in Rasmussen's encephalitis. Ann Neurol. 2007 Jul;62(1):67-80.).

good luck!
jan
Title: Re: Anti-caspase 3 for apoptosis in brain
Post by: ImmunoNYC on January 24, 2009, 10:34:15 PM
I used the anti-cleaved caspase 3 from Cell Signaling in paraffin sections of many tissues from mice and humans with great success. Use spleen or tonsil as positive control. Very specific assuming your protocol is good.
Title: Re: Anti-caspase 3 for apoptosis in brain
Post by: mol7 on September 08, 2010, 07:46:07 AM
Hi,
Can someone please recommend a non rabbit anti caspase 3 antibody to detect apoptosis?  I will be carrying out IHC-fr. I cannot use rabbit since I plan to co localize apoptotic cells with specific antigens and most of my antibodies are anti rabbit.
Thanks in advance