Week 6

I turned in a proposal to my mentor to describe the specifics of my experiment and what materials I would need (tissues, primary and secondary antibodies, serum) so he can get them and I can get started. I will only be able to work with a control tissue (no AD) and severe AD because the other intermediate tissues are ROS (religious orders study) tissue, which is tissue from older religious clergies. When they use this tissue they can make sure the results aren't disturbed by other activities that harm the body, like drinking and smoking.
I've also decided that instead of also doing a western blot, I will do a fluorescent IHC. There are two types of IHCs and I will be doing chromogenic IHC for the first part of my project (which will show where the tangles and plaques each are) and then I will do fluorescent IHC for the second part (which will show where the tangles and plaques are together in the same sample).
I did a lot more research this week and started working on my presentation.

Description of Project

This is a description of my project (I know it's a little late but better late than never).

Alzheimer’s is a progressive disease that causes dementia and destroys other important behavioral functions. There is no cure for Alzheimer’s yet and many of the details, such as the cause of the disease, are uncertain. However, research shows that neurofibrillary tangles, caused by hyperphosphorylation of the tau protein, and amyloid plaques, caused by beta amyloid accumulation, are key causes of neuronal death. In my project, I will be looking at the concentration of tangles and plaques in the frontal cortex and the hippocampus. 

I will be keeping my title (Tangles and Plaques in Alzheimer's).

I won't be doing western blots anymore. Instead, I will be doing another type of immunohistochemistry (Fluorescent IHC).

Week 5

Not much happened this week. I made a couple solutions and changed the title of my project, but I didn't run any IHCs. I talked to my mentor and hopefully I will start my own project next week or the week after. Right now I am working on a proposal where I will write up the things I need for the experiment (primary and secondary antibodies, animal serum, and tissues) and what I plan to do so that my mentor can get my supplies ready.

Pictures

These are the pictures that will hopefully explain what I talked about last week.

This is how the tissue looks at the beginning. It's a little hard to see but the floating white things in each well are the tissue samples. Since I was mounting at the very beginning they were not separated in wells (they were just in one tray). Also, this tissue is human brain tissue which is bigger than the rat brain pieces, but I just wanted to show you how it looked before mounting.

After I smoothed out each piece from the jumble (top picture), I had to put it in the order shown on this paper.

This is an example of the final. Some of the pieces were ripped so it made it even more difficult than it already was to match it to the picture above because sometimes a cut off piece could look like it was from the top row, but it turned out that it was a piece of another part.

Week 4

This week I did two new things.

Brain sectioning: We had a part of a brain about the size of the palm of a hand, and we needed to slice it into pieces 40 microns thick (which is EXTREMELY thin) so that tests can be done on those. First we had to freeze the brain sample about halfway by placing it on top of a block of dry ice. We do it half way so that we can freeze the rest gradually so there aren't chatter lines when we cut through it with a microtome. After that we used sucrose as a glue to stick the brain to the holding plate on the microtome. We surrounded the brain by powdered dry ice. We set the microtome to 40 microns and began cutting when the brain was frozen enough (if you run your hand over it and it feels like your gliding your hand on glass, then you're good to go). The process took an extremely long time because our sample was about an inch thick and we had to cut it into such small pieces.

We were doing IHC on a TBI rat brain and we had to mount the tissue onto slides. The experience was different then when I had to mount human tissue because I had multiple tissues to mount on each slide, they were so small, and they needed to be put in a certain order. First I had to scoop the brain tissue into a container filled with PB and sort through all the tissue (there were about 15 pieces each). A paper showed the different sections of a rat brain (if it was cut vertically) and I had to sort through all the pieces and put it in that order. It was extremely difficult because the pieces were really small and some were ripped. After that I had to individually mount them onto the same slide. I didn't take pictures but maybe when I go back I can take them because it sounds really confusing in words.

I plan on starting my own experiment either 2 or 3 weeks from now (next week I won't be going because of spring break).