Programmed cell death (or apoptosis) is an essential cellular process in which unneeded, aged, and hazardous cells are deleted from eukaryotic tissues. However, overactive apoptosis in the brain causes neurodegeneration and is linked to a number of diseases such as Alzheimer’s and Multiple Sclerosis. Several factors trigger programmed cell death in neural tissue, including excitotoxic insult via high incidences of neurotransmitter receptor activation, such as that which occurs with AMPA. Studies have shown a relationship between excessive AMPA excitation in the hippocampus, and excitotoxic cell death.
Apoptosis is induced by a family of cell death receptors and their ligands. Cell death signals are transduced by death domain containing adapter molecules and members of the caspase family of proteases. These death signals finally cause the degeneration of chromosomal DNA by activated DNAse. The mouse DNAse that causes DNA fragmentation was identified recently and designated as CAD (caspase activated deoxyribonuclease). The human homolog of mouse CAD was more recently identified by three separate groups independently of each other, and is thus termed CPAN, DFF 40, and human CAD. These three terms are now used interchangeably.
DFF 45/ICAD is the inhibitory protein of DFF 40/CAD and forms a complex with the DFF 40/CAD. Upon cleavage of DFF 45/ICAD by activated caspase 3, DFF 40/CAD is released and activated, allowing it to enter the cytosol then into the nucleus where it eventually causes DNA degradation, which is the hallmark of apoptotic cell death. CAD is a caspase dependent endonuclease.
In our lab, we use Chemicon International’s Rabbit Anti DFF 40/CAD Polyclonal Antibody (catalog #AB 16926) to confirm the presence or absence of CAD in the cytosol and nucleus of AMPA insulted pyramidal neurons using laser scanning confocal microscopy. We use Chemicon’s CAD Antibody to perform immunohistochemistry on hippocampal tissues of 8-15 day old Sprague-Dawley rats. We have been performing a temporal assessment of the presence or absence of CAD after the AMPA insult in both the CA1 and CA3 regions of the hippocampus.
This Anti DFF 40/CAD Antibody crossreacts with human, rat, and mouse tissue. The antibody is recommended for use in Western blots at a concentration of 1:500 – 1:1000. After performing a dilution curve we found 1:250 to be an optimal concentration for our immunohistochemistry studies. We detect CAD binding using a goat anti rabbit Alexa 488 secondary antibody from Molecular Probes for our fluorescent confocal microscopy analysis.
We have been using Chemicon International’s Rabbit Anti DFF 40/CAD Polyclonal Antibody for over three years now and have been extremely pleased with it. It has given us relatively consistent results. We have found Chemicon’s Rabbit Anti CAD Antibody to give a relatively strong signal in the hippocampal CA1 and CA3 pyramidal neurons. We chose this CAD antibody because it was the one we found that cross reacts with our species of interest (rats). Most others only cross reacted with human and sometimes mice. This is a relatively inexpensive antibody compared to many other antibodies for apoptosis studies. It also can be stored for up to 12 months in a standard 4oC refrigerator, as opposed to other antibodies which are only good for 3 – 6 months and/or must be stored at -20oC. I would definitely recommend using Chemicon International’s Rabbit Anti DFF 40/CAD Polyclonal Antibody to anyone who is doing apoptotic studies.
VelvetLee Finckbone, MS
Lab Technician
Texas Tech University Health Science Center