Programmed cell death (or apoptosis) is an essential cellular process in which unneeded, aged, and hazardous cells are deleted from eukaryotic tissues. However, overactive cell death 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 characterized by several morphological nuclear changes, including chromatin condensation and nuclear fragmentation. These changes are triggered by the activation of members of the caspase family, CAD, and other novel proteins. One such product recently identified, Apoptosis Inducing Factor (AIF), causes chromatin condensation and DNA fragmentation, both of which are hallmarks of apoptosis. AIF is a caspase independent endonuclease. It is localized in the mitochondria and released into the cytosol and translocates to the nucleus when apoptosis is induced. This causes the mitochondria to release apoptotic proteins, cytochrome C, and caspase 9. AIF is highly conserved between humans and mice, and is widely expressed.
In our lab, we use Chemicon International’s Rabbit Anti AIF Polyclonal Antibody (catalog #AB 16501) to confirm the presence or absence of AIF in the nuclei of AMPA insulted pyramidal neurons. We use this 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 AIF after the AMPA insult in both the CA1 and CA3 regions of the hippocampus.
This particular AIF antibody cross reacts with human, rat, and mouse tissue and is recommended for use in Western blots or for immunohistochemistry using formalin fixed, paraffin embedded tissues (using an antigen retrieval method). Although we do perform immunohistochemistry, our tissues are not paraffin embedded and we do not use an antigen retrieval method. After performing a dilution curve, we found 1:100 to be an optimal concentration for our immunohistochemistry studies.
We detect the AIF antibody using a goat anti-rabbit Alexa 488 secondary antibody from Molecular Probes, then perform laser scanning confocal microscopy. In our hands, we have found Chemicon’s Rabbit Anti AIF Antibody to give a weak to mild signal in the hippocampal pyramidal neurons.
We have been using Chemicon's anti-AIF antibody for over three years now and have been somewhat pleased with it. It has given us relatively consistent results. We chose this AIF antibody because it was the one we found to cross react with our species of interest (rats). Most others only cross react with human and sometimes mice. This antibody is relatively inexpensive compared to many other apoptosis antibodies. 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. With the signal being so mild, eye strain can sometimes be an issue, although this could probably be compensated for by using a higher concentration. I would recommend (with some reservations due to the weak signal) using Chemicon International’s Rabbit Anti AIF Polyclonal Antibody to anyone who is conducting apoptotic studies.
VelvetLee Finckbone, MS
Lab Technician
Texas Tech University Health Science Center