Week 8: University of Chicago

August 20th, 2013

Rachel Henning ’15, Becker Fellow in Research

It’s crazy to say that this is my last blog! I’ve had an amazing experience this summer!!! This weekend I got to meet up with Sharon, a recent Cornell graduate! We went to Navy Pier and the view from the top of the Ferris Wheel was amazing!!!
sharon and me
She couldn’t believe that I hadn’t had a Chicago hot dog yet so we went to Portillo’s! The food was so good!

The beginning of this week I spent most of my time working on my oral presentation and some time converting all of our confocal images into a format that will enable us to quantify the intensity of the quantum dots, which would give us an idea of differences between cell types in the amount of exosomes that were taken up. This came from the comparing non-enriched confocal images with environmentally enriched images. The image on the left is of non-enriched exosomes and the image on the right is of environmentally enriched exosomes. As you can see there is some differences in the intensity/amount of exosomes that were taken up.
NE GFAP EE GFAP

Tuesday evening my friends and I sent out for sushi. I had never had it before and I don’t know if I will ever have it again :)
sushi

Wednesday was the big day! I presented my research at the 2013 Chicago Research Forum and Poster Session. I was a bit nervous for the presentation, but my lab was super awesome and let me do a couple run-throughs with them ahead of time.
poster me and poster

Here is my poster for my summer research. It turned out very well I think! It was great a experience having to prepare a scientific research presentation and poster. Below is Dr. McDade who is the person who created the PSOMER program I was a part of this summer.
dr. mcdade and me

Thursday was my last day working in the lab for this summer. I worked on actually quantifying the images I formatted earlier this week. I didn’t get to finish it, but the results I did get were very interesting!
Friday I had to move out of the dorms and say goodbye to everyone. I made some amazing friends this summer and had an amazing experience! It was crazy to think that 8 weeks ago my suitemates and I were complete strangers and now are great friends!
suitemates

Turning my keys for the lab in and saying goodbye to everyone in the lab was hard! They were great mentors for me this summer and taught me so much!
lab

Week 7: University of Chicago

August 13th, 2013

Rachel Henning ’15, Becker Fellow in Research

This weekend was pretty awesome!
On Saturday my suitemate Chelsey and I went to Wicker Park and explored a street festival there. We found a photo booth and had some fun!
Photo booth

I got to shadow Dr. McDade, who is an anesthesiologist, at the University of Chicago Hospital on Sunday. I got to watch him instruct a resident on how to do two epidurals. He also explained everything as he went which was super interesting! But the coolest thing was when he did a spinal anesthesia/spinal block for a woman who was pregnant with identical twins. After that, I was able to watch the entire caesarean section from start to finish! It was amazing to get to see the two baby girls be born! The twins were monochorionic-diamniotic, meaning that they shared the same placenta but had their own amniotic sac.

Since our staining was working we proceeded to do the second half of slices for our staining on Monday.

Tuesday we had another confocal appointment to image the slices we stained Monday.
EE CNPase red EE CNPase green EE CNPase overlay
We noticed something really interesting about the staining for the oligodendrocytes. The exosomes, which are shown in the red image, outlined the oligodendrocytes (which are shown in the green image). At first we didn’t notice this because as you can see in the 3rd image, which is an overlay of the red and green images, the red outline isn’t distinct. So we didn’t count many positive cells (positive cells=cells that contained the exosomes) at first. However, once we noticed the red outlines, we determined that those were also positive cells.

On Wednesday we went back over the images from the 1st confocal appointment to see if there were any red outlines in those images as well and recounted the number of positive cells. After we recounted the images we got to finally perform statistical analysis on the data. I was super nervous that we would have done all that work and maybe not have found statistical significance. Luckily I was wrong and we found that environmentally enriched exosomes trafficked preferentially to oligodendrocytes in comparison to non-environmentally enriched exosomes!!!! So exciting!

Thursday and Friday I worked on putting all of my data and images into graphs and figures for my oral presentation next week. So many little details to work out that I would never have thought of before, like not using Excel to make my graphs and having to do all the lines and stuff in a new software program.

Week 6: University of Chicago

August 6th, 2013

Rachel Henning ’15, Becker Fellow in Research

Through my friends on the volleyball team here at the University of Chicago I met a group of students who play sand volleyball! So Saturday of this past weekend I went up to North Avenue Beach and played sand volleyball for a good portion of the day! It was so much fun and made me realize how excited I am for this upcoming Cornell College Volleyball season!

We were bound and determined to fix our staining! We had narrowed down the possibilities a bit last week and all we could do was keep plugging away at the remaining possibilities.

Monday was crazy! We looked at slices from each step of the slice culture fixation, blocking, and staining. When we were getting blobs everywhere we then made all new solutions for everything in case one had gotten contaminated. Even with all new solutions we kept seeing blobs!!! Even slices straight out of the incubator had the blobs!

Monday evening I finally went into the dome library to work on my paper. It is a pretty cool set up being able to see the sky and have natural light while studying!
20130722_200724

Tuesday was the big day!!!! We investigated each step in the slice culture incubation process to see how we could have something fluorescing before we had started staining it. We discovered that the Sytox® had gotten contaminated or gone bad. The Sytox® was used to check that there wasn’t cell death in the slices before we used them for staining. After this discovery we tried a different Sytox®, Sytox® Orange. The old Sytox® is the green image below. The new Sytox® Orange is the clear image! No more blobs!!!!!
sytox 6.3x green for blog
sytox 6.3x orange for blog

After weeks of not being able to stain without blobs appearing, Wednesday we finally got to get back into staining!! Since staining for oligodendrocytes doesn’t require letting it go overnight, we tried that on Wednesday. The slices turned out amazing!!! No more blobs!!! The image below is of a good oligodendrocyte stain I did, blob free!!!
cnpase for blog

I met with Darrell Nabers on Wednesday, who works in the Pritzker Medical School Admissions Department here at the University of Chicago! I sent him a draft of my personal statement thus far and he gave me great feedback and talked with me one-on-one about applying for medical school! It was a great meeting!

Thursday morning my program had another group meeting, during which Dr. McDade, who used to be a part of admissions at the Pritzker School of Medicine, broke down how to write a good personal statement paragraph by paragraph. This program has been such a wealth of knowledge for me concerning the application process for medical school!

In the lab on Thursday I worked on taking slice cultures what had non-enriched exosomes and staining for microglia, astrocytes, oligodendrocytes, and neurons! Friday we had a confocal appointment to see some results finally!

Friday seemed to go so slowly in the morning! I was so excited to go to our confocal appointment to see how our blob-free staining turned out!!! I wasn’t disappointed!
confocal NE microglia
The top left image is of the tagged non-enriched exosomes. The top right image is of our microglia staining. And the bottom left image is an overlay of the top two images, which showed us exactly where in the slice cultures the exosomes went!! So neat!!!!!

Week 5: University of Chicago

July 30th, 2013

Rachel Henning ’15, Becker Fellow in Research

This past weekend my family came to visit me in Chicago! Of course we went to Giordano’s for deep dish pizza!
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We also went to a Cubs vs. Cardinals game. Sadly the Cardinals lost :(
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Taste of Chicago was going on so we stopped by there before they drove back to Iowa. Although it seems like the 1st 4 weeks flew by, it has been hard not being able to see my family.
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So on Monday we had another confocal appointment. Our Q-dots weren’t as bright as we would have liked, but we could still see them. However, these mysterious blob-like cells were stained green on our slides!!! That made it hard to see the cells we really were concerned about.

Tuesday we did a couple stains for microglia and olidodendrocytes without any Q-dots. We used the microscope in our lab to look at the slides and the blobs were there again! We thought maybe something had happened to our secondary antibody that stains green.

To check if our green secondary antibody was the problem on Wednesday we did two different microglia stains, one red and one green.
green microglia
red microglia
In both the red and green stains the blobs were present! Thus our secondary antibody isn’t our problem.

We thought maybe something was wrong with the primary antibodies for microglia and oligodendrocytes! So we started prepping some slices to stain for neurons and astrocytes since those require different primary antibodies.

On Thursday I finished the staining for astrocytes and neurons. The neurons were stained well but there were more cells stained than should be, probably those dreaded blobs again!
green neun

astrocytes
The astrocytes were stained green but those blobs make the image a bit fuzzy.

I also went to a mentor lunch on Thursday. It was helpful to hear what criteria the medical student based his medical school choice on.

Our new secondary antibodies came on Friday! We tried the staining for neurons and astrocytes with the new secondary antibodies and guess what! The blobs were still there! Below is a picture of the stain for neurons.
neun better

We went back to the drawing board and systematically went through each part of the staining to see where the blobs first appeared. We were able to narrow it down quite a bit so hopefully next week we will figure it out and be able to stain again!

Even though things haven’t quite been working out as planned, I still learned a lot this week! Trying to problem-solve the staining issue definitely involved a lot of critical thinking! I feel like we are very close to solving the staining problem and hopefully this time next week I will be getting blob-free stains!

Week 4: University of Chicago

July 22nd, 2013

Rachel Henning ’15, Becker Fellow in Research

I got to visit a high school friend of mine over the 4th of July weekend!!! It was nice to escape the city for a few days and relax in nature!!!
court and I

On Monday I ran another BCA protein assay to compare the protein concentrations of unstimulated and stimulated exosomal protein from dendritic cells. My results definitely improved from the last time I ran A BCA protein assay. I also fixed some slice cultures with methanol which tends to make better imaging. However, I went to mount the slices onto a slide I learned that the methanol dehydrates the slices a bit and they were very tricky for me to mount! So my job is to figure out which cells these exosomes, which are only like 100 nm, are traveling to. Partially since the exosomes are so small I tag the exosomes with QD-CD63, which fluoresce red, and I stain certain cells within the slice cultures. I then look at the slices under a microscope and see which cells the red dots went into. The QD-CD63 are super sensitive to light so I have to keep the slices in the dark as much as possible so I hide the slices under a box.
20130711_114852

Tuesday I stained for oligodendrocytes and when I looked under the microscope it appeared that the exosomes had entered into the oligodendrocytes. But since the microscope in the lab can’t look at different planes, it is hard to tell if the exosomes are in the oligodendrocytes or just the surface. The only way to know for sure is to go to the confocal microscope, which can look at the cells from all different planes. But my staining worked which is always a good thing!
Our standards for the BCA protein assay were getting low so I got to make new ones. As you can see in the picture, the intensity of the purple varies, which is due to variations in protein concentration. My new standards are the lower two rows and the old standards are the upper two rows.
20130709_141956

Wednesday I stained some slice cultures for microglia and put some tagged exosomes in! This is the final product!
microglia
So the green stuff is microglia cells and the red are the tagged exosomes. The orange and yellow areas are places where the exosomes went into the microglia! Success!!!!

On Thursday my program has it arranged that we get to have lunch with mentor students. The mentor students are students who are either in medical school, graduate school, or both! We got to chat with them and ask any questions we had! It was a great opportunity to hear a bit more about the Pritzker School of Medicine here at the University of Chicago. Apparently here you don’t receive grades your first 2 years of medical school! Interesting! I also got to learn how to do an SDS-page gel, which separates proteins based on their size. I was then able to take the separated proteins from the SDS-page gel and transfer them to a western blot.

On Friday I took the western blot from Thursday and tagged Alix proteins, which if present will help us confirm that we have exosomal protein. We had a shorter day because there was a BBQ for everyone who’s here over the summer that evening. It was great to get to talk to other summer program students as well as doctors and researchers.

Week 3: University of Chicago

July 15th, 2013

Rachel Henning ’15, Becker Fellow in Research

Due to the 4th of July holiday this was a little bit of a shorter week.
Over the weekend I got to meet up with some Chicago Cornellians, Melinda and Millie, and see the city a little bit! Here we are visiting the “Bean” in Millennium Park!
bean

On Monday I got to do a practice run of the procedure I will be doing for the research. We are curious as to which cells the exosomes are going to and if they have a preference for a certain cell. So I introduced red-tagged exosomes to the slice cultures and then tagged the oligodendrocytes (a type of cell in the brain) in the slice cultures green. To do all of this I had to first bind primary antibodies to the oligodendrocytes and then bind the secondary to the primary antibody. The secondary fluoresces green and allows us to see where the oligodendrocytes are. These images only show the oligodendrocytes. It had been too long since the exosomes had been tagged red and the red fluorescence had diminished.
STAINS OF OLIGO 3 (3)
STAINS OF OLIGO 2 (3)

I also ran a gel to check the purity of the conjugated QD-CD63 population that we isolated last week.
pic for post
This image suggests that our population is basically pure.
Kae and I then made two batches of exosomes and QD-CD63 and mixed them in order for the QD-CD63 to tag the exosomes.

Tuesday was a short day due to our weekly lab meeting in the morning. I worked with data analysis in the afternoon. I went for a run after lab with my friend Jazlyn. Here is us at Promontory Point!
980293_10201054453037117_95189501_o (2)

On the way back to our dorm we found Cornell Ave!
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On Wednesday Kae and I took one of the gels we ran last week and cut out the band of conjugated QD-CD63 that we isolated. We then placed the band in a dialysis tube and used electrophoresis to separate the QD-CD63 from the gel.

At the beginning of the week I had a meeting with all the other students in my program and we got to learn about the application process for medical school from the University of Chicago’s Deputy Provost, Dr. McDade. Dr. McDade actually started the PSOMER program back in 2007. In 2012 the average MCAT score for the incoming class at Pritzker School of Medicine at the University of Chicago was 36! It’s pretty cool to hear what he has to say since he helps with the medical school interview process here at the University of Chicago!

Week 2: University of Chicago

July 2nd, 2013

Rachel Henning ’15, Becker Fellow in Research

Monday of week two was great! First off we worked on de-fatting the cerebellum slides. Being as fat is hydrophobic, it gets in the way of the luxol fast blue staining the myelin. We placed the slides in the luxol fast blue dye overnight. Then I got to watch Aya and Kae harvest bone marrow cells! The RPMI media I made last week was actually used to harvest the cells! We only want the bone marrow cells so to get rid of the red blood cells we added red blood cell lysis buffer to cause the red blood cells to burst. After watching it a few times I even got to add lysis buffer and centrifuge the cells myself.
After we removed as many of the red blood cells as we could, we removed a sample of the bone marrow cells and looked at them under a microscope. I actually got to count the bone marrow cells so we could get an estimate of how many cells we had harvested. Made me remember my cell culture dishes from BIO 141! We harvested approximately 240 million bone marrow cells!

Tuesday I got to continue with the cerebellum slides. I prepared one of the solutions for the luxol fast blue stain. Luxol fast blue is a staining technique that hasn’t been used much in this lab so I got to kind of experiment with it to figure out what would produce the best results. This is what the finished product looked like.
luxol fast blue pic
Aya was working on cell culturing so I got to observe that first hand! For that she was primarily concerned with the hippocampus, which was sliced super thin and placed in wells. This allows us to have cells that can be manipulated as need be and after a few weeks these cells are basically the same as cells in an adult rat.
Kae and I then worked with Q-dots, which basically are these tiny nanoparticles that can bind to other particles of interest and can fluoresce lots of different colors!

Wednesday on the way to work I saw this person in the rain.
2013062695104213 (2)
Once in the lab, I got to learn how to make electrophoresis gels to run antibodies on. I loaded in a control Q-dot sample and the Q-dot sample Kae and I made on Tuesday which we conjugated to an antibody called CD63. This is the gel, the separated band tells me that the Q-dots conjugated to the antibodies did bind because the control on the left containing unconjugated Q-dots only had one band and the Q-dots conjugated with antibodies had two bands. The top band is the Q-dots that did not conjugate to CD63 and the bottom band is the population of Q-dots that did bind to CD63.
QD_QD-CD63_altered and resaved
I also got to learn how to do a BCA protein assay, which I found out will be pretty important to my project! I had samples of unknown protein concentrations so I had to use a set of standards to then be able to compare the unknowns to, which that part was similar in concept to a couple of my analytical chemistry labs at Cornell.

Thursday I ran another gel but this time we cut out the part of the gel that contained the extra band. This band is our conjugated antibodies and Q-dots. We used a special filter to extract conjugated antibodies and Q-dots from the gel. Now we have our pure population of Q-dot-CD63 ready to attach to our exosomes!

Friday we used the conjugated antibodies and Q-dots to tag the exosomes. Once we tagged the exosomes, we introduced the tagged exosomes to the slice cultures. The Q-dots fluoresce, so when we looked at the slice cultures under the microscope we could see where all of the antibodies traveled by the red dots! The red dots inside the cell mean that our tagged exosomes were taken in by the cells in the culture slices.

Then I got to look at the luxol fast blue slides I had been working on all week under a microscope! The dark blue areas are white matter and the light blue areas are grey matter.
LUXOL FAST BLUE 2 and resaved

Most of the other students in my program have either already taken the MCATs or are currently in the process of studying for the MCATs. They have been super great about offering me advice concerning my own preparation for my MCATs as well as sharing some of their practice materials. All of the other students in the PSOMER program are either going to be a senior this upcoming year or are working towards their masters. It is nice having another year to prepare my medical school application and I will be able to ask any of the PSOMER students for advice.

This week I got a lot of hands-on learning. Seeing how everything is done and why certain things are done has helped me get a better grasp on our project. It’s strange to think that I’ve already been here two weeks already! I keep learning new things everyday, which has been awesome!!

Week 1: University of Chicago

June 21st, 2013

Rachel Henning ’15, Becker Fellow in Research

So I’ve completely settled into Chicago! Monday I moved in, met Dr. Kraig, and met the other students in my program. Almost everyone in my program plans on applying for medical school or medical and PhD programs.
smaller
The building with the pink windows is my dorm and the statue marks where the first isolation of plutonium occurred and where the world’s first nuclear reactor was made. It was made on a squash court under the old college football field.
Tuesday was orientation day and the students in the Pritzker School of Medicine Education in Research (PSOMER) program got to get to know each other better, tour the undergraduate campus and tour the medical school campus. We also were given our first white coats!
20130618_171531
Wednesday is when the real fun began! I got to see the lab and meet everyone who works there. Dr. Kraig is a Cornell College alumni! He has his MD in Neurology and his PhD in Physiology and Biophysics. He focuses on neurological disease and on how environmental enrichment (increased social, physical, and intellectual activity) can lead to an increased resistance in the brain to neurological diseases. He is the principle investigator at the lab I’m working in this summer. Kae Pusic is a postdoctoral scholar who uses her knowledge of vaccine development and delivery to work on developing immune based-therapies for brain injuries and migraines. Aya Pusic is working on her PhD in Neurobiology at the University of Chicago. Aya focuses on how T-cells immune signaling can cause spreading depression and she also focuses on the relationship between increased environmental enrichment and decreased vulnerability for spreading depression. Yelena Grinberg is also working towards her PhD in Neurobiology. She focuses her research on peripheral signaling factors part in cognitive decline due to aging and disease. Here’s the link to the lab’s website, they research some pretty interesting topics http://kraiglab.uchicago.edu/ !
smaller sign
I became more informed as to what my project would be this summer and I am super excited about it! We believe that environmental enrichment leads to an increase in myelination, which leads to an increase in cognition. Thus an aged brain can become young again by enrichment!- Our lab is defining the naturally occurring processes responsible. I even got to help measure the integrated optical density of pixels of images of gray matter! Of course school is never over so I’ve been reading up on exosomes, gray matter, white matter, environmental enrichment, multiple sclerosis, and neurobiology in general! We even came up with a working title for the paper we are planning to write based on the research this summer: “Environmental Enrichment-Induced Exosomal MicroRNAs Involved in Oxidant Defense and Therefore, Myelination of Aging Brain.”

One place I still need to visit is the undergraduate library! It has a dome for reading in!!!
smaller dome
I got to do more hands-on work Thursday, I sliced the cortex of rats brains and mounted the slices to microscope slides for myelin staining we will do next week. It was so neat to see the different parts of the brain!
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I also helped measure the integrated optical density of white matter in environmentally enriched brains vs. non-environmentally enriched brains.

My experience as a lab assistant at Cornell came in handy Friday when I made RPMI media for the dendritic cell cultures.
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Later in the day I got to go to see Yelena use confocal microscopy for her research project.
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I learned that the green are astrocytes, the red are nuclear factor kappa-B (which are cytosolic transcription factors that be be ‘activated’ to move to the nucleus of cells), and the blue are DAPI (labeling DNA, thus the nuclei of all cells in the brain tissue slice).

I’ve been planning on attending medical school for quite awhile now, but I think this fellowship might sway me to apply for MD/PhD programs so I can conduct my own research as well as see patients.

Well that’s all for this week!!!

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