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