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Neuron Counting Procedure

Procedure using Immunocytochemistry:

Following the experimental trials, use a standard protocol to isolate the maternal rat brains and freeze them prior to further sectioning. Once ready, section the brain on trim mode until ideal brain regions are identified. Utilize a brain atlas to ensure proper sectioning of the hypothalamus, amygdala, central gyrus, and any region the instructor desires to analyze. For our experiment, these specified regions are important in maternal rat behavior, because each region has certain functions such as memorization, special learning, behavioral patterns, and emotional behavior, all of which are important when testing for maternal rat behavior.

Once the brain is completely sectioned, carefully place each section in a 30-well dish with approximately 300 microliters of PBS. Ensure that all material and supplies needed are accounted for as well as proper safety regulations are being followed such as clovers, lab coat, and glasses. Next, wash the brain sections with PBS 3-5 times then place on the shaker for 5 minutes after each wash. Next, add the primary antibody to each well and store in the fridge overnight. That following day, wash the sections again with PBS prior to adding the secondary antibody to each well. After the secondary antibody has been on rocker for 10 minutes, wash with PBS 3 more times and add Avidin-Biotic Complex or ABC which aids in the neurological connection between each neuron. Finally, add DAB which is a mutagen to our cells, so extra precaution and safety must be taken as always in science labs. Lastly, wash brains with a final PBS and store in 20 degrees Celsius in fridge until ready to place on microscope slides. When creating the wet mound slides, place a brain section on the slide and drop distilled water and alcohol to remove foldings and allow for the brain to spread out across slide. Also, utilize a microscope brush to add in the process. Once on the slide, remove the water and alcohol and place a cover-slip over section. Store the slide in a slide box for further analysis.

Now that the slides are ready to be analyzed, set up the microscope, camera, and computer to start recording the data. Here, Q Capture Paint, and Image J software is used to count the number and size of the neurons in a specified brain region determined by the research instructor.