Week 5 in the lab saw four big developments:
1. Finishing NeuN immunochemistry and moving to GFAP: NeuN immunochemistry is where you stain mice brains (at sizes of 50 micrometers) to view neuron channels under a microscope. Then, my mentor Yuling can run analyses on the brains in order to compare the amount of neurons between wild-type and genetically modified mice. I have been doing this type of immunochemistry for the past two weeks, but starting next week I am moving onto GFAP, which will stain brain slices to view glial filaments. The process between NeuN and GFAP immunochemistry is almost identical, however you use different secondary antibodies that bind to the primary.
2. Cutting brain slices: In order to start running GFAP, we need brain slices to stain. My mentor taught me how to use the machine that slices the brain slices to your desired thickness (which in our case is 50 micrometers). I spent about two hours cutting over 100 slices from one brain, which we then froze in a -80C freezer until we start the immuno next week.
3. Performing perfusion on three mice: Whole animal perfusion is a way to kill an animal and then fix it with paraformaldehyde to obtain the best preservation of the brain. This is an important process, because it needs to be done correctly in order to obtain brains that are preserved well and can be worked with either for staining or western blots. To perfuse a mouse, you first sedate it with ketamine threw a intramuscular injection. Once the mouse is fully under anesthesia, you pin it down stomach up in order to cut into the stomach. After cutting through the stomach you go up through the sternum to expose the heart. You inject a butterfly needle into the heart and pump in saline, and then quickly cut the heart to kill the animal. The saline is taken up through the capillaries and flushes out all of the blood. Once the animals lungs are white, and there is no longer any blood left in its system, you switch out the saline for paraformaldehyde, which is a fixative that preserves the brain instantly.
Here are some pictures from the procedure.
4. Joining a behavioral studies project: At lab meeting, my PI Dr. Siegel announced that he needed people to help in a behavioral study that tested Dextras1's effect on memory, EEG readings, and locomotion. I am specifically working on running 30 mice through a T Maze, a paradigm that tests working memory. I run the mice through 8 trials and then analyze the results.
Week 2
This week brought about another new project, as well as the start of my behavioral testing,
1. GFAP: This week I ran immuno for GFAP on brain's, and it went very well. My slices were all in tact with no breakage, which gave my PI the confidence to give me a project....
2. PROJECT: I got a project I am working on with one other high school student Meghan for the duration of my stay. I am using GFAP immuno staining to analyze if ketamine exposure in adolescent mice produces any differences in glial filaments when compared to wild mice. This may not seem related to schizophrenia, the labs main focus, however schizophrenia is a NMDA antagonist and is thought to have a correlation to the disease. I will start immuno next week for this project.
3. Behavioral testing: I ran my first trials on all 30 mice this week in the T maze. It took me the full day, but it was actually pretty fun. This was the first time I got to interact with the mice completely alone, which made me nervous at first but in the end it was fine. Next week I will have two more testing days before I can start analysis.
3. Clinical trials with my PI: Dr. Siegel not only runs a lab, but he meets with patients every Wednesday afternoon at UPenn's hospital. I was able to shadow him for the whole afternoon. Unfortunately, I cannot divulge much information because of HIPAA, but it was a great experience and I got to actually meet people dealing with schizophrenia. It was a really moving experience and made me glad that I was working in a lab that was trying to help people like them live a normal life again.
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