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Jane Richardson, It’s Art, Not Science

My lab centers their research around the study of Giardia, a parasite that infects the small intestine. Giardiasis, the disease caused by Giardia, often leads to Irritable Bowel Syndrome (IBS). In the late stages of Giardia and in IBS, high levels of serotonin (5-HTT) are present. We are confident that this is what links the two diseases together. Our first step in answering all of our questions is determining what causes high levels of serotonin in Giardia. One theory we have is that high expression and presence of the serotonin transport protein (SERT) causes higher levels of 5-HTT. To do this, we are trying to stain difference cross sections of small intestine of infected and non-infected mice to determine the changes in levels. To do this, we must use the art of IHC. Immunohistochemistry (IHC) is about finding the perfect blend of antibodies, chemical washes, bead baths, and various other factors to produce the result you want. Over my two weeks here, I have been told constantly that it’s more art than science. Essentially, we have to first use non-specific antibodies, that will bind to all the proteins that have general functions in the cell. Then, we use a primary antibody to adhere to the SERT protein. Then, we use an additional secondary antibody that has a fluorescent glow to attach to the primary antibody. Then, we can see exactly where in the cell the SERT is. Although this may seem like a simple task, finding the correct balance of these antibodies is very difficult. The staining cannot be too dull, but it should not have too much noise. Over the past two weeks, my grad student and I have been trying different combinations of IHC protocols to try to maximize our procedure. We have tried different antibodies and even used swiss-roll slides from the mouse, instead of mere cross sections. We are still working to maximize our protocol. At the same time, we have been make cDNA from the RNA in our cells so we can use them for rt-PCR. cDNA is DNA that is made using reverse transcriptase from the RNA in the cell. This allows you to figure out which genes the cells when transcribing at the time of cell death. When we use real time polymerase chain reaction (rt-PCR), we are able to see exactly how much RNA was in the cell at the time of death. This will help us determine the differences in gene transcription in healthy and infected cells at different times of infection, specifically related to 5-HTT. First, we isolate the RNA, use the nanospectrometer to measure RNA concentration, create our master mixes, and put our RNA in the thermal cycler to convert them to cDNA.

I have really enjoyed working at my lab thus far. I was extremely nervous at first, but as I have gotten more acclimated to the lab setting, I am really enjoying myself. I am no getting more control over my project and more freedom to try new things. I only expect this to grow in the future. Also, I will be able to make my on media before I leave, which I am really looking forward to. My lab loved the donuts I brought in! They are some of the nicest people I have had the pleasure of knowing and I excited for my next 5 weeks!
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