Since my last progress report, I have learned a great deal and become more self-sufficient. I have achieved relative independence in the mouse room. This means that I can wean mice (i.e. separate the pups from the mother while simultaneously separating the boys from the girls once they are about twenty-eight days old) by myself and maintain records of what I do. This has also made me realize that maintaining a healthy and well-documented mouse colony is vital to most of the science we do. I have also largely accomplished both my goals.
While I have not completed the entire qPCR process from start to finish (my first goal), I have completed every part of it separately. I am currently still in the process of isolating the RNA because it took longer than anticipated. This is in large part because of the quantity of samples I am using; I am isolating RNA from the spleens, spinal cords and brains of 12 different mice with varying genotypes. As mentioned in my first report, once I have completed the isolation I will convert the RNA to cDNA and run a qPCR to determine expression levels. In regards to my second goal (completing an immunohistochemistry), I have accomplished it in full.
An immunohistochemistry is a technique that researchers use to stain different parts of tissue or types of cells differently. For example, we use it to stain and visualize different components, cell types or proteins in brain tissue. We stain microglia in one color, apoptotic cells in another and so on, using a microscope to see each staining. The staining is achieved by using two sets of antibodies. The first set of antibodies, the primary antibodies, is designed to recognize and bind to antigens of specific elements, i.e. microglia or apoptotic cells. The second set of antibodies, the secondary antibodies, is designed or chosen so that they will recognize and bind to only one of the types of antibodies from the first set. The secondary antibodies are also coupled with a specific fluorophore such that when a specific wavelength of light is shined on the sample, it will fluoresce as a result. Fluorescence is when a photon of light hits a sample and is emitted at a longer wavelength, for example, when blue light is shined on a sample and green is emitted. So, in this way, each individual element will emit a different wavelength based on the fluorophore and the antibodies used.
My primary goals for the next couple of weeks are improving on and perfecting what I have already learned. For example, I would like to improve the efficiency with which I isolate RNA. The homogenization of the tissue in particular is difficult and something I can work on. I would also like to be able to use theconfocal microscope independently because I am currently learning how to work it. It is a delicate piece of equipment that you have to handle carefully. My final goal is to be able to collect the brain, spinal cord and spleen from mice while maintaining the integrity of the organs. This is important so that when we use them downstream for various applications we can get accurate results.