To recap my previous blogs, my project over the summer was an attempt to find a way to fluorescently stain the neurons that we record from in the lab. The issues that we ran into were that it was very difficult to create a stain that would allow us to record from a live cell that was also stable enough to last through the fixation process. After doing some research on possible ways to stain neurons, I finally decided that the best way to solve our problem was to attempt to stain the cytoskeleton.
The cytoskeleton is essentially the backbone of the cell. It is a dynamic structure composed of three separate types of filaments: microfilaments, intermediate filaments, and microtubules. These filaments lend the cell structural support as well as aiding in cell division, organelle movement, and cell motility. Of the three types of filaments, I decided that the microtubules were the most likely candidate for staining. Microtubules are the largest of the filaments, and they are located throughout the cell and are integral to cell structure. As a result, staining the microtubules would be a fantastic way to view the entire cell’s morphology.
There are several ways to stain microtubules, but ultimately I decided that the method that would best suit our purposes was to use a flurescently labeled tubulin that would integrate into the microtubule structure. Microtubules are formed by the combination of two types of tubulin subunits, known as alpha- and beta- tubulin. Thus, injecting fluorescently labeled tubulin subunits into the cell would result in the incorporation of the tubulin into the cytoskeleton. If this procedure worked it would be perfect for what we are attempting. It would allow us to view the cells that we recorded from using a fluorescent microscope, plus it would be a lasting stain since the tubulin is completely integrated into the cytoskeleton. This would allow us to perform FISH on the cells while still being able to keep track of the cells we recorded from.
After telling my professor about the plan, he was all for it. We ordered the fluorescently labeled tubulin and we began to perform experiments with it. Unfortunately, this procedure has never been done before, so a lot of this has been guesswork. After several tries with one concentration of tubulin, we haven’t been able to label a cell, but we are hoping that it is simply a matter of creating more concentrated amounts of tubulin to inject in the cells so that it is properly incorporated. Unfortunately, we ran out of time in the summer to attempt the experiment with a higher concentration of tubulin, but I look forward to trying again once school starts up, and I will be sure to keep everyone posted on the future results.