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

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