1-21-2010: More dissections...

Today I and Dr. Nuss did a lot of dissections, but these were different from the previous two types of dissections we have done so far. For these dissections, we isolated the midgut of the mosquito and will test on a western blott to see if a phosphorylated insulin receptor is present in the midgut of bloodfed mosquitos. The results will be compared to the peptides we find in non-bloodfed mosquitos. We expect to see a lot of phosphorylated insulin receptor in bloodfed mosquitos because our hypothesis is that bloodfeeding starts the insulin pathway. We believe that proteins from the blood interact with a receptor in the midgut (which may or may not be present... we will see) which then sends a signal to the brain and causes neurohormones to be released and sent to the ovaries of the mosquito which signals egg production and ultimately controls the reproduction of the mosquito. So, in order to isolate the midgut in order to test for the phosphorylated receptor, we put about 100 mosquitos on ice in order to anethesize them and then decapitated them so that we could pull the midgut out of the mosquito from the tail (part of the midgut is attached to the head of the mosquito which is the reason for the decapitation). By pulling on the bottom tip of the abdomen of the mosquito, the outer layer rips and the attached ovaries, digestive tract, midgut, and salivary glands all come out in one connected string of organs. We then seperate the rest of the organs from the midgut and then put the midgut into solution (made up of ingredients that preserve the midgut and inhibit proteases from degrading our target peptide). We had to isolate the midgut from 40 bloodfed and 40 non-bloodfed mosquitos, so we did that and then prepared the extraction solution. After setting the midguts into dissection solution, centrifuged them and then resuspended them into homogeniztion solution (and I am not sure what the function of this solution is). We then ground up the pellet into the solution in order to release more of the target peptide. We then stored our samples in the -80 freezer.

In the midst of doing dissections, I also prepared the western blott for the primary antibody wash. I put the antibodies on and the nitrocellulose paper is soaking in the antibodies overnight.

Tomorrow I will not be shadowing Dr. Harper because I found out that I have to have a background check and a TB test in order to shadow in the operating room. However, I will be shadowing an OBGYN, Dr. Larrimore, tomorrow.