In the week before (Week 3), Dr. Sinakevitch and I had taken 5 bees and injected the fluorescent dye into specific areas of their brains, notably, the antennal lobes and the projection neurons, so that these areas would show up in confocal imaging. During Week 4, I continued my practice of this injection procedure, specifically, the surgery needed to open the bee's head for injections. For my Week 4 practice, I took 2 new bees, one on Monday and one on Tuesday. Unfortunately, when I practiced on those two bees, I failed the surgery both times. I accidentally pushed my razor too far into the head for one of the bees, and I failed to make the proper incisions in the other bee's head, meaning I couldn't close that bee's head again. The locations of the incisions are hard to see, and the bee brain is a very delicate structure - I needed more practice and experience before I could get this procedure down. My first-time success in Week 3 was beginner's luck, I guess. Good thing too - Dr. Sinakevitch and I were able to use that lucky bee brain for imaging, alongside the four other good ones Dr. Sinakevitch had performed surgery on then, so we were still able to use all 5 bees from Week 3 to get proper results. During Week 4, while I practiced the injection procedures, Dr. Sinakevitch did the immunohistochemistry to label the GABAa receptors in our Week 3 samples, and we were set to image.
When we imaged the brains from Week 3 with confocal microscopy that week, we got results. Dr. Sinakevitch had used immunohistochemistry to label the GABAa receptors with antibodies after we had done the injections. Due to both the fluorescent antibodies and the fluorescent dye, we were able to light up the parts of the brain we cared about with one color, and the locations of the GABAa receptors within those parts showed up as another color. With this procedure, we were able to deduce that the GABAa receptors were not located in the olfactory receptor neurons, which are in the antennal lobes. This result was the main result we communicated in the deliverable poster we made for the 2017 Arizona Imaging and Microanalysis Society's Microscopy Conference, which was that Friday. I spent the last day of Week 4 assisting Dr. Sinakevitch in editing the poster, and I even got to show her some tricks on Microsoft Word while we were writing parts of our poster. Unfortunately, I couldn't go on Friday - I was in UT Austin at the time for a college orientation. My editing did help a ton for the poster though, as my mentor is not completely fluent in the English language and needs someone to help with her grammar. I was also very proud that my only surgical success amounted to something, and relieved that I could understand what was going on. And I was super happy to be second author on that poster - that poster is hanging now on the walls of ASU's ISTB (Interdisciplinary Science and Technology Building) 1.
Week 5 was when I finally got to see the immunohistochemical side of our neuroanatomical experiments. Of course, I also kept practicing the surgical injection procedure that comes before. In Week 4, though we had gotten results from the olfactory receptor neurons, we had not gotten results from the projection neurons - we were not able to use the images. Monday of Week 5, we took five bees, this time, to image them for results from the projection neurons. Dr. Sinakevitch opened the head and injected for three bees, and left me two to practice with. Very generous.
"I couldn't fail this time," I thought as I got the materials ready. My thoughts didn't matter - pretty soon, I lost another bee, and I was left with just one more.
I proceeded as carefully as I could with that bee, and this time, I succeeded in injecting and closing the head again. The bee had survived the procedure. How relieved I was! I gave Dr. Sinakevitch that bee so she would have another brain to use alongside her three. The next day, however, I was informed that my bee died shortly after I gave it to her. We were down to three bees.
Depressing, but this is where it begins to get better. Dr. Sinakevitch had gotten her three brains out, and she decided it was time I learned the immunohistochemical procedure. I learned how to prepare the agarose gel and how to block our brains in the agarose, before learning how to use the vibratome to take sections of our brains. I got to take sections for two of the three brains, and I got it perfectly. It came easy - much better than the surgery. Next. Dr. Sinakevitch showed me the procedure of washing the sections, to prepare them for the primary antibodies. This means applying the PBS (phosphate buffer solution), with the detergent in it, to the sections at least 6 times. I did this part too. Time management was crucial for this step, because taking out and putting the solution had to be timed, but luckily, I've always liked being punctual. After the first application of the solution for one hour, the next applications had to be for 10 minutes each. There are other ways to do this timing, but Dr. Sinakevitch said this timing was okay. Dr. Sinakevitch came in after that and applied the primary antibodies, and our sections were ready. There are steps after this, which I was supposed to see on Wednesday, but I fell sick on Wednesday with a flu, and I couldn't come in. But it's okay - I still have videos of me doing the washing and the vibratome sections. I also got pics of our poster, and the confocal images I saw.
My thoughts? Much of this is still quite complicated. I am still adapting to the lab. Despite this, however, I am learning so much, maybe more than I ever have before, and I am already making my impact. And I'm doing okay - I can do the immunohistochemical steps, and I think now, I can succeed when I do the surgery again. I almost succeeded on that last bee I performed surgery on - next time, I know it'll be better. I am confident I can go forth in neuroscience and neurobiology after this, carrying new abilities and experiences from this lab that will definitely come in handy.
Hi Jaeyoung! It's great to see your progress every week and I look forward to seeing what you ultimately accomplish!
ReplyDeleteFor someone like me who is not as fascinated by science as you are, how do you suggest entering into the realm of cutting-edge and complex technical scientific work? I can appreciate the big-picture conclusions that arise out of these experiments, but I can't seem to get into the details of the instruments. Any advice?
Thank you!
Hello Anna! It always starts with reaching out to labs - reach out to enough college labs, and at least one will take you. Usually, they will be willing to guide you through the complex procedures and explain the little details. Knowing everything you learned from AP Bio and your bio textbooks really helps too.
ReplyDeleteI see. So experience will be be my best science teacher, and exposure will be the best technology advocate! Thanks for your response :).
ReplyDeleteHey Jaeyoung!
ReplyDeleteThe work you are doing is quite interesting. You mentioned in the post that your lab used the five bees from week 3 and determined the GABAa receptors were not located in the olfactory receptor neurons. Is having a sample size of only five bees enough to prove what you deduced is true of the entire bee population? I remember from our stats class together it was either a minimum of 10 or 30 depending on what you were doing. Is it possible since you are dealing with anatomy that the minimum sample size rule does not apply?
Thank you!
Hi Nikash!
ReplyDeleteTechnically, the sample size is pretty large because what we were sampling were the receptors in each brain, and there are a LOT of them (much more than 30), even in one bee brain.
I'm sampling receptors for where they're located, to answer the question of which cells have/don't have the receptors, and I sampled enough receptors to say "the receptors aren't primarily located in the olfactory receptor neurons."
ReplyDeleteAny more surgeries performed recently? I can't imagine how difficult and challenging it must be to have to operate on the brain of a bee! What type of equipment do you have available to you? Can you post some pictures of your results or do you need to wait until they are more conclusive?
ReplyDeleteWe haven't done surgeries lately - it's been more molecular biology, with RNA Isolation and RTq-PCR. We may return to surgeries next month. I talked a bit about the equipment we use last time - we use a scalpel and a (sort of) gripping device to break off a small part of the scalpel for use. We use Ringer's to clean up during surgeries, heating rods to heat the Ecosane to seal up the area again. Then, there's the current injection device we use to actually do the injections, which uses an electrode to essentially launch drops of neurobiotin dye into the brain. When I present, I'll go through these procedures in more detail.
ReplyDeleteI do have some pics of our neuroanatomical results - will be posting soon. Tonight, I'll be making a post about how it's related to the molecular biology and genetic work I'm doing now.