Peddie EXP Summer 2018

For my final week at my lab, I began by continuing my work with antibodies and immunofluorescence. Because we have not seen any positive results with SERT, we have moved on to looking at other antibodies. This week, I began working with the protein CCK. CCK, or cholecystokinin, is responsible for initiating digestion in the small intestine. On the round of CCK that we did, we got some very promising results in the disparity of infected and uninfected slides. On my last day at my lab, my PI came back from his conference in London and took our whole lab out to lunch. We got to hear about all of the new scientific discoveries that were discussed at his conference, which was extremely interesting and we talked a lot about new projects that the lab could explore. Everyone in my lab was so nice this summer and I really appreciated them taking the time to take me out to lunch. We went to a deli in Georgetown and ate on top of the Car Barn, which is part of the Georgetown business school. ...

These two weeks, I decided to keep myself open to new opportunities so I could get the most out of your lab experience. Though I have been working primarily with SERT in staining slides, I got to try several new things this week that I am really excited about. First of all, I began working with another protein (tryptophan hydroxylase) that produce 5-HTT in an attempt to explain the high levels of serotonin in Giardiasis and IBS. One interesting thing that came from this slide production was that though SERT levels are higher on the outside of the intestine, trp levels are found to be higher on the inside of the intestine. This was very surprising to me because I assumed the reverse would be true. As for my continued work with SERT, levels in the distal colon still seem to remain higher than any part of the small intestine. This was an unexpected result that we first thought was a fluke, but now is presenting itself more and more. We will continue to look into that as we keep analyzin...

In science, sometimes the only way we can actually reach our destination is with all of the hidden failures along the way that guide us to the end game. I guarantee every scientist will experience their fair amount of failure, but all of it will make you better. In optimization, essentially the entirety of my job, this is especially prevalent. When figuring out the best procedure, you have to go through a lot of failed ones. In addition to the one successful procedure and the one in progress, we now have yet another to optimize. Last week, we started a procedure to extract bacterial DNA from human stool samples. We ran a gel to see if the DNA extraction was successful. Our extraction procedure was utterly unsuccessful. Though it would have been nice to find success on the first try (something I now realize was wishful thinking) it is going to make the end result much more of a success. Without stumbles along the way, we can never get better. In continuation with our overall proje...

We finally did it: an optimized IHC protocol. Peering into the microscope to see the bright red below was an amazing feeling. The SERT was glowing like a Christmas tree. Our procedure incorporated two new elements that helped us achieve the finished procedure: pre-heating of the slide and antigen retrieval. Before beginning the slide washes, we heat the slide in a 55 o C water bath. This makes the slide more susceptible to the treatments we use. To complete antigen retrieval, we place the slides in .1 M citrate buffer and then heat it, using a 100 o C water bath. This exposes epitopes that were covered in the making of the slide. Therefore, the tissue is more susceptible to antigens. With these two new steps, we were finally able to see the SERT, in Cy3, and the background staining, in DAPI. However, one pitfall of our microscope is we can only look at one contrast at a time. This forces us to take separate pictures of each contrast and overlay them in photo editing (picture of inf...

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 t...