First Week

My first week has been great.
I have learned the protocols for what I need to do to successfully determine if the organoids are growing properly. The organoids are stored in a huge freezer because they are cryogenically frozen in a gel that is a clear liquid at room temperature (this is super important). It is so cold that it has a little sign on the side of the door that says that you should not touch anything inside with unprotected hands. The shelf and containers used to keep the freezer organized is always covered in a layer of snow. I have to pull out a heavy metal container and hold it as close to the freezer as possible to prevent the contents from melting. After removing the appropriate box, I then quickly run over to the sectioning machine. The holding chamber is kept at -16 degrees Celsius. There are many different types of organoids. For example, some are electrocuted with a certain voltage a certain number of times. Each little cube also has organoids of a certain age. Some harvested from the culture at 28 days others are only harvested after 200 days or more (they are frozen after they are harvested).

First, I have to section the organoids into fine slices. This allows me to look at a very small layer of tissue. The holding chamber contains an apparatus that allows me to cut up to 1 micrometer slices. For the purposes of the experiment, I only need to slice with a width of 20 microns (micrometers). That is about as thick or even thinner than half the thickness of a piece of tissue. I turn a crank to move the block along the edge of a really fine blade (I have been told multiple times not to let my hands near it). The consistency of the block is about as think as slicing through butter. After I make a slice, I then transfer it onto a slide that has been kept at room temperature and is labeled appropriately. You may be asking how I transfer it over without tearing the sample. Remember I said that "they are cryogenically frozen in a gel that is a clear liquid at room temperature" was super important? That's right! We flip over the slide and hover it near the sample. The sample will catch onto the slide and melt right on (literally the coolest thing ever :) ). The gel is clear and dries because it is so thin. The dried gel traps the organoid slice onto the slide but allows for fluids and particles to act on the tissue. I then store the slides in the fridge overnight. After intense sectioning this Monday and Tuesday, I can finish one block in 45 minutes. This includes the time for cleaning the chamber, mounting the block onto a chuck, aligning the block against the blade so that the slices are even, slicing the blocks and mounting the slices onto the slides (10-12 per block). Each slide has around 4-8 samples. In total, for one block, I make at a minimum 40 slices. Usually, it is around 72 slices, though. I finished all of the sectionings that I can do with the organoids that they have today so I won't be sectioning anymore.

The next part is antibody staining. Although I understand it theoretically, I've only seen my supervisor perform it once so I don't know the specific protocol in-side-out. There are six different stains that they use. Off the top of my head (the lab notebook can't leave the lab so I don't have my notes in front of me), five are used to show that the cells are undergoing mitosis (division) and one is used to show that cells are differentiating. This proves that the cells are growing, dividing, and differentiating. The type of antibody stain is labeled after the protein that the first antibody (FA) attaches to. The FA is programmed to see a specific protein as a threat and attaches to it. In a normal immune system, once the antibody is attached to the substance, it would trigger more antibodies to bind and also signal a macrophage to come and eat itself along with the foreign object. Obviously, there are no macrophages so the FA is just stuck onto the protein. The goal of antibody staining is to attach a fluorescent protein onto cells with a specific protein available in the cell. The second antibody (SA) is the one that has the fluorescent protein attached. The SA sees the FA and deems that protein as a foreign object. Therefore, just like the antibodies in your body, it attaches to the FA. While this process can identify if a process in the cell is occurring, it can only produce qualitative data, not quantitative (what is more respected in the scientific community). I will be showing my supervisor the process so that she can check and make sure I am not missing anything.