Glorified Deli Meat Slicer | #3

Besides mounting rat brain slices on slides, I have also been cutting the rat brains using a machine called a microtome-cryostat.

The microtome-cryostat in the Flagel Lab. Going from left to right, the containers on top of the cryostat contain 100% ethanol, OCT compound, microtome blades, Kimwipes, and lubricant.

As the title of this blog summarizes, a microtome-cryostat is basically a refrigerated deli meat slicer. “Microtome” refers to the actual cutting machinery and “cryostat” refers to the refrigeration the machine provides during the cutting process (-23°C in the picture above). Because brains are so delicate, even after fixation with formaldehyde and immersion in sucrose solutions, making precise cross-sectional cuts of the brain means that the brain has to undergo further processing. We typically roll up tubes of aluminum foil (with one closed end), cut off the cerebellum at the base of the brain (to make a stable foundation for the brain to stand upright and also because the cerebellum isn’t a region of interest), place the brain cerebellum-side down, and then fill the tubes with OCT compound. OCT compound stands for optimal cutting temperature compound and it is used to embed tissue samples prior to frozen sectioning on a microtome-cryostat. Since OCT is a viscous liquid at room temperature, upon topping off the aluminum tubes with OCT we place them in dry ice to quickly freeze them. Conventional freezing techniques aren’t sufficient because they are too slow and ice crystals that can damage the tissue will form. After this step, the brains are ready for cutting in the microtome-cryostat.

Diagram of the microtome-cryostat.

To operate the cryostat, we peel the aluminum foil off the now solid tube of OCT. Next, we obtain a pre-cooled platform and apply more OCT on the base. Before it freezes, we quickly put the solid tube of OCT onto the platform+semi-frozen OCT so that it can stick on. After that,  we can secure it on the oscillating part of the microtome-cryostat which is operated by the hand wheel. Consistent and precise cuts in the micrometer (μm) range can be made by adjusting the settings in the machine and then turning the hand wheel in a clockwise motion. This is probably somewhat hard to envision without a detailed diagram but for the most part, it operates in the same way as the deli meat slicer in your local butcher shop. The only difference is that the slicer, in this case, is in a refrigerated enclosure and the cuts can be thinner than a human hair.

TL;DR: refrigerated deli meat slicer that cuts frozen brains. 

We can then collect the slices of interest in well-plates filled with cryoprotectant where they can undergo even further processing via radioimmunoassays or immunohistochemistry (IHC). These techniques allow for us to identify and locate proteins of interest using specific binding agents called antibodies. From the pictures below, you can see an example of brain tissue in a well plate that has undergone IHC.

Brains that have undergone IHC using an Anti-mCherry antibody. m-Cherry is a protein that can be selectively expressed in different brain regions and running an IHC allows us to track where it was expressed.

Hopefully you haven’t been overwhelmed by the scientific lingo so far, sometimes I get carried away describing the finer details in a scientific procedure (it comes second nature haha). But to address the prompts, being in Ann Arbor is definitely a change in pace from my hometown of Elmhurst, NY. Elmhurst is located in the borough of Queens in New York City and as part of the “city that never sleeps”, a million different things are happening at all hours. Amazing restaurants, specialty supermarkets, and all manner of things are either a short walk or train ride away in NYC. Ann Arbor is somewhat similar in that regard, with many resources largely accessible on foot. However, the pace of the town is definitely slower, especially since a large part of its identity revolves around the University of Michigan. To elaborate, fewer kids are in school during the summer and this definitely results in a significant mellowing out the atmosphere. Regardless, it’s nice to be on a college campus over the summer because there are so many great study spots to get your work done. Real estate is limited in NYC, and quality public study spaces are sparse.

In regards to mentoring in the lab, the postdoc I’ve been working with, as well as my lab manager, have been great sources of guidance throughout this opportunity. That isn’t to discredit my PI by any means but typically, the PI in a lab doesn’t spend much of his/her time in the lab. Instead, you would find them taking care of the bigger picture, guiding research plans, applying for grants, and steering the boat in the right direction—a visionary in a sense. The rest of us do the work that is needed to produce the results. That being said, most of my work has generally been assigned by my PI but more specifically ordered by more experienced members of the lab like the postdoc and lab manager. To clarify further, my PI creates a rough outline of what I should be working towards and the postdoc/lab manager help manage what I do on a day-to-day basis to build towards that end goal. With weeks of continuous mentoring, I’ve started to develop more autonomy in the lab and it’s been great to see the assignment of increasingly more involved tasks especially as I obtain and master new skills. Everyone in the lab, not just the people I’ve been working directly under, have been tremendously receptive and helpful whenever I have lingering doubts when completing a task or need an extra hand with running a procedure. All in all, it’s been a pleasure working in the lab so far—not only is the research interesting but more importantly, the people I work with are so great to be around.

Until next time,

Jason Wong

One thought on “Glorified Deli Meat Slicer | #3

  • July 24, 2018 at 9:15 am
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    No worries about the scientific lingo, Jason! It’s really fascinating to hear about what you’re doing in your internship.

    It can really feel great to see that your supervisors are trusting you more and more by seeing that your tasks are increasing in complexity. It sounds like you’re really getting the hang of this research! I know for myself that when I tell someone else about how to do something, it increases my own understanding of how to do it, so maybe writing out what you’re doing in the lab in your blog posts has helped you to solidify your knowledge.

    I’m so glad to hear that you are finding some good mentors in the postdoc and lab manager. What have you learned from them?

    Looking forward to reading your next posts and learning more!
    Maggie

    Reply

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