c-fos Staining and Immunohistochemistry
c-Fos
is an immediate early gene (IEG) that codes for a transcription factor
that is thought to mediate long-term changes in neural functioning. Once
the c-Fos protein is formed it interacts with another IEG product,
jun, to create a heterodimeric transcription factor that regulates the
expression of “downstream” late response genes. Increased c-Fos
expression is induced by a novel experience, such as a rat being introduced
to a new cage, receiving an aversive foot shock, or, as is the case in
this lab, being administered their first intraperitoneal injection of
epinephrine.
Ninety minutes after the rats receive an injection of 0.3-mg/kg epinephrine,
they are anesthetized and debrained. The brains are then sliced into 50
mm thick sections using a vibratome. Once the sections are prepared, immunohistochemistry
techniques are used in order to quantify the amount of c-Fos protein
induced by the epinephrine injection. We use an avidin-biotin method in
order to stain the brains for the Fos. To begin the tissue is incubated
with primary antibody, rabbit anti rat c-Fos, for two days. After
this the sections are then treated with biotinylated goat-anti rat secondary
antibodies. Following this incubation the sections are treated with the
Avidin-Biotin complex, which binds tightly to the secondary antibodies.
Finally, the sections are stained using a solution that contains the staining
agent DAB.
Once the tissues are stained they are mounted on slides and viewed under
a microscope. The desired images from the slide are then captured and
the Fos proteins counted using NIH Image 1.61.
The goal of this experiment is to provide further support for previous
findings in this lab that have suggested a path by which epinephrine exerts
its effects on the brain. In the proposed pathway the epinephrine excites
receptors on organs that provide projections to the nodose ganglion, which
then synapses onto the nucleus of solitary tract (NTS) via the vagus nerve.
Neural projections from the NTS may excite the locus coeruleus, the amygdala,
and the hippocampus, all areas that are known to be important in memory.
Cedric
Williams
The University of Virginia
Psychology Department