Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.
The overall goal of this immunohistochemical detection against c-FOS protein is to assess the neuronal activity in brain tissues. This method can help answer our key questions in the neurophysological field such as brain mapping and identification of brain regions involved in specific physiological regulations. The main advantage of this technique is that c-FOS gene has a low expression in the absence of stimulation, which allows easier quantification of neuronal activity in our test simulation.
To begin this procedure, wash the coronal brain stem sections for 10 minutes with 0.1 molar PBS at room temperature. Repeat the wash three times. Next, suppress the endogenous peroxidase activity by incubating the sections with 3%hydrogen peroxide in 0.1 molar PBS for 30 minutes at room temperature.
Then, wash the sections for 10 minutes with 0.1 molar PBS at room temperature and repeat three times. Block nonspecific binding sites by incubating the coronal sections with 2%goat serum in PBST for one hour at room temperature. Subsequently, incubate the sections with a rapid polyclonal antibody against the c-FOS protein in PBST with 0.25%bovine serum albumin for 48 hours at four degrees Celsius.
In this step, wash the sections with 0.1 molar PBS for 10 minutes at room temperature and repeat the procedure three times. Next, incubate the coronal sections for one hour at room temperature with a biotinylated goat anti-rabbit antibody in PBST with 0.25%bovine serum albumin. After one hour, wash the sections for 10 minutes with PBST at room temperature and repeat three times.
Then, incubate the coronal sections for one hour with an avidin biotin peroxidase complex in PBST. Subsequently, wash the sections with PBST at room temperature for 10 minutes and repeat two more times. Then, wash the sections for 10 minutes with 05 molar Tris buffer at room temperature and repeat the wash two more times.
Next, incubate the coronal sections with DAB, nickel ammonium sulfate, and hydrogen peroxide in 05 molar Tris buffer at room temperature. Add 17 microliters of hydrogen peroxide to the remaining Tris buffer. When the staining intensity is optimal, stop the reaction by washing the sections with 0.1 molar PBS for 10 minutes at room temperature, and repeat the wash four times.
Then, wash the sections with distilled water. Now, mount the sections serially on the slides by carefully spreading them in rostrocaudal order on the slides with a brush. After that, let the sections air dry and clearly label the slides according to the samples.
Then, dehydrate the sections by immersing the slides in an absolute alcohol bath for 30 seconds at room temperature, and repeat the procedure twice. Next, immerse the slides in a xylene bath for three minutes at room temperature and repeat twice. Subsequently, apply five drops of mounting medium on each slide.
Place cover glass on them and avoid forming air bubbles. Let the slides air dry for 48 hours. Shown here is a drawing of a section of the medula oblangata.
The dotted area shows the commissural and median parts of the nucleus tractus solitarius. The rectangle indicates the region that photomicrographs were taken. These are the photomicrographs of c/mNTS in the condition of normoxia or under hypoxia.
The black arrows show c-FOS-positive cells. Here is a histogram showing the mean number of c-FOS-positive cells per section in c/mNTS in the condition of normoxia or under hypoxia. The number of c-FOS-positive cells was significantly higher in the condition of hypoxia than that of normoxia.
After watching this video, you should have a good understanding of how to use the c-FOS immunohistochemical detection as a marker of central pathway involved in specific physiological stimulation.
