Week 10

This was my last week. I only went on Tuesday. I spent about 2 hours taking pictures of my results. There is a microscope attached to a computer which we can take pictures from. I got some good results and I can't wait to show them in my presentation.

Week 9 and Protocols

This week I did a Fluorescent Immunohistochemistry. It's basically the same procedure except the secondary antibody is fluorescent, the primary antibody is a lot more concentrated, the tissues must be kept in the dark after the secondary is added, and a few other things. I made a mistake during the experiment. Generally, we can put two antibodies together but only if they are made in two different animals. I was targeting tau and amyloid proteins so I put both their antibodies in. After 2-3 hours I realized that both antibodies were made in mouse (so I wasn't supposed to put them in together because then the secondary could bind to either and it would not differentiate) so I washed the tissue as much as I could in hopes that the antibody hadn't blinded yet. Then I put only one antibody in for amyloid plaques. Hopefully the results are okay. I briefly looked at them under the microscope and it seemed fine.

Week 8

I cover slipped my tissue this week:
     Steps for cover slipping:

•      Dehydrate the slides by putting them in 2 solutions of 70% ethanol, 2 solutions of 95%      ethanol, and 2 solutions of 100% ethanol for 5 minutes each
•      Put the slides in 3 solutions of xylene for 5 minutes each (xylene is in the fume hood)
•      Take a slide out of the last xylene with forceps and wipe the back
•      Smear a line of DPX along the slide and carefully slip the cover on the slide
•      Use the back of the forceps to push bubbles out 

Do one slide at a time.

I also completed a successful Nissl stain, and will work on analyzing that next week.

Week 7

I started my experiment. Everything flowed nicely and it was a successful experiment. Unfortunately, the person who gave me the tissue accidentally gave me two control tissues from the hippocampus instead of one control and one severe AD which means that I can't compare the tangles and plaques in control and SAD in the hippocampus. I will instead do a Nissl staining which will show the different sections of the hippocampus. I might be able to get actual SAD tissue from the hippocampus (but mostly likely not) and then I'll do the immuno on it if I get it.

These are my results from the IHC I did this week.

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).

Week 3

Like I expected, I had to change my plan again. Apparently the lab doesn't have that much Brain Trauma tissue, so I can't do that. I have to change my blog title but I haven't come up with anything yet so I will do that later. I'm sorry to disappoint the people interested in the TBI part of my project. I worked it out with my adviser and this is my new plan (that will hopefully be my last plan):

I will be doing IHC on a regular brain, a mild Alzheimer's brain, an Alzheimer's brain, and a                    severe Alzheimer's brain. I will be looking for the neurofibrillary tangles (tau protein) and the amyloid plaques (amyloid protein) and hopefully I will see an increasing amount as I go from least severe to most severe Alzheimer's cases.

I plan to also do western blots which will be the second part of my experiment. IHC shows where the protein that I'm looking for is and if there is any, while western blots show how much of the protein is present in the sample.

So this week was pretty uneventful. I didn't do that much because I usually shadow someone doing an experiment and help out, but we were waiting on some tissue samples so we only ran a couple experiments.

Second Week

My weekly schedule is about the same- I go to the lab three days a week and do immunohistochemistry (IHC) on samples of brain tissue. This week I attended a conference on amyloid which is a protein in the membrane. In Alzheimer's patients, the amyloid breaks off and forms plaques outside the cell that disrupts synaptic signaling.

What I Learned: There are two types of samples you can do IHC on: floating tissue and mounted tissue. Last week, I watched/helped someone do IHC on floating tissue, and this week I did it on mounted tissue. There are a few differences in the protocol- (for mounted tissue) the tissue is mounted onto slides at the very beginning of the process and is rehydrated and put in the oven before the regular procedure starts. Also, we use a special marker, that keeps liquid from crossing over, to draw a circle around our samples so that the primary antibodies actually affect the sample (rather than floating onto the surrounding slide).

I'm not 100% sure what to analyze from my IHC yet. After we get a successful IHC, we analyze it. For example, we can look for which areas the protein concentration is highest, or whether or not the protein is even there, etc.
I have a few ideas:

• Do IHC on a regular (without Alzheimer's) brain tissue sample for tau and a separate IHC for amyloid. Then do IHC on Alzheimer's brain tissue sample for tau and a separate IHC for amyloid. Compare. Then do IHC on brain trauma tissue for tau and a separate IHC for amyloid and compare with Alzheimer's and regular tissue.
• There are five stages of brain trauma tissue that we have starting from Stage 1 (which is the least severe) till Stage 5 (which is the most severe). Do IHC on tissue from each stage for tau and amyloid and note differences. Do IHC on Alzheimer's and compare all the results. (I'm leaning toward this one more).

I might not do either of them because as I learn more, I change my plan (this would be the third time I've changed my plan in the last 2 weeks).

Forgot to say this...

I forgot to mention that my project is turning out to be very different from what I thought it would be so I might have to change the title...

First Week

I've never been to a professional lab so I had no idea what to expect but I realized right when I came to my lab that my project is not actually what I thought it would be. The first difference was that I thought the lab was solely focused on determining the similarities and differences between traumatic brain injury and Alzheimer's, but we actually look at a bunch of different diseases too, like Parkinson's and Down's. The second difference was that I thought we would all be doing one big experiment but be doing different parts. What I mean is I thought it would be more like the Chemistry catalase labs we've been doing where one of the team members could make the color reagent while the other could set up the water in the tubes. Everyone here works on their own experiment. It all contributes to the Alzheimer's research, but everyone does their own thing.

Things I Learned: I learned a lot of things like how to use lab equipment, like microtomes, how to check the pH of something (not with the litmus paper, with like an actual machine), and about the different procedures they run. There are two procedures, western blot and immunohistochemistry (IHC), that I am focusing on for my project. The purpose of IHC is to find a protein in a tissue sample (in this case a brain tissue sample). We are specifically looking for the tau protein because usually tau stabilizes the microtubules in the axon so that the neurotransmitters can flow more stably down the neuron, but in Alzheimer's, hyperphosphorylation of the tau occurs so it detaches from its position and floats around. The tau is sticky so it sticks to each other forming a tangled mess (neurofibrillary tangle) that prevents things from flowing through the neuron leading to its death. 

I haven't really researched the western blot yet because I'm first going to make sure I understand all the details in the IHC protocol because apparently the western blot is more difficult.

What I Did: This week I mostly did research. I shadowed a person doing IHC and did a few buffer washes for her (part of IHC protocol). 

Days I Went: Monday, Wednesday, Thursday