Copyright © 2020 Tanvi Banota
I can’t believe summer is already halfway over! And that I’m only going to be in the lab for 4 more weeks :( But regardless, 4 weeks is better than none; I’m super excited for the next few weeks where we’ll really start working on compiling all our research together for our paper and poster and so forth. As for this week, we did a lot of prep with our final presentation in mind. Lots and lots and lots of IHC. And flow cytometry. And protein assays. But mostly IHC. So much. And along with all of that, there was so much failure this week. Nothing we did this week was a success. It was “You get a failure! You get a failure! Everyone gets a failure and complete waste of time!” Sigh…
But the week started off really interesting. There were takedowns Monday, Tuesday, and Wednesday, and each day a portion of the mice were analyzed for pulmonary function before the lungs were removed. We got to see the mice get hooked up to a ventilator machine called a FlexiVent, that would regulate pressure, volume, and frequency to retrieve information about lung function for each mouse being sacked. This day really stretched my conscience though; in order for the FlexiVent to properly gather data, the lungs still need to be able to take in air and breathe, so the mice had to be technically alive. So the people doing the takedown had to subjectively decide when the mice were effectively anesthetized but not euthanized, and sometimes you could clearly see the mice were still alive, twitching a little as we gave them tracheotomies and the cannula was inserted into their trachea. …I dunno, it got to me. But anyways, that aside, it was incredibly cool to see the mice hooked up to the machine and see the readings and the differences between control and treatment groups. And we found a radio in the room so we were jamming out to 90s Greatest Hits on some random station during the takedowns.
Right after the takedown, I helped Alexa with the flow cytometry preparation of the BAL (bronchoalveolar lavage) fluid. We went through the long process of centrifuging and aspirating and lysing literally 5 times. (You really need to make sure only the right cells are in that sample.) We made up the antibody cocktail, with 7 antibodies that each cell would be screened with and then detected for using flow cytometry. Sadly, I had to leave before I could actually see the flow in action, but it’s a relatively simple process - it uses the fluorescently tagged antibodies and a laser to detect which color is expressed by which cell. It was all really interesting, except it was not even for my project, so it’s not like I’ll be able to use any of the data. That’s another thing about science - you spend half of your time helping other people with their projects.
The coordinators of the Partners in Science program also made their site visits at the beginning of this week, so we got to talk to them about our projects and experience and everything and give them a tour of our labs and generally show them around.
On Tuesday, we spent literally the entire day doing day 1 of IHC for the iNOS antibody. This is a classic M1 macrophage pro-inflammatory marker, and something we could use for pictures for our poster. But oh my god a word of advice. Never ever ever do IHC on more than 10 slides. Since our iNOS IHC worked out really well last week with that concentration, we thought we might as well stain all the slides we needed to do with iNOS in one go. 20 slides. Never again. It was torture. Trying to keep track of all the slides, making sure the tissue was facing the right way, all those washes! Impossible. And oh my god do not even get me started on how long it took to coverslip all of those slides on Wednesday. Pure torture. We spent literally all of Wednesday on day 2 of IHC because of the sheer number of slides. And the worst part is, it didn’t even turn out that great! It was actually stained pretty badly, at least compared to last week’s iNOS IHC, and we have no idea why. We literally did everything the same, but I bet we messed something up because of the amount of slides we had to manage. Ugh. I hate that part of science, messing up and not being able to fix it. But anyway, we showed Alexa, she said we’ll scan the slides and count the cells and depending on the numbers we’ll go forward from there. We should probably be able to find a good spot for pictures for our poster, so it won’t be a complete waste. But ugh, I hate to think that we wasted those 2 days and more importantly, all those slides.
On Thursday, we were supposed to score those slides from goodness knows how long ago, but again, the grad student who was supposed to teach us wasn’t coming in, so we started another round of IHC. We needed some more markers for more data points for our papers/poster, so we decided to mass troubleshoot, as in, try 4 different antibodies at the supposed correct concentration to see if any of them worked. We did two different types of antibodies, recruitment markers and M2 anti-inflammatory markers. We used CCR2 and CX3CR1 for our recruitment markers - these stain for macrophages that are traveling to the tissue, the infiltrating macrophages that have yet to specialize into M1 or M2 (pro or anti-inflammatory) but are clear signs of inflammation. And we used CD68 and CD163 for our M2 markers, staining for macrophages that are specifically anti-inflammatory, helping the tissue heal and combating the possible detrimental effects of M1 macrophages. Anyway, those were the antibodies we planned to do IHC with. Of course, nothing goes according to plan. Ever. It took us more than an hour to even find one of these antibodies, the CCR2 one, and that one was like 2 years old. After scouring some more, we found CD68 and CD163 antibodies that were even older. We couldn’t find CX3CR1 at all (apparently a lot of these antibodies had been thrown away when the freezers went down a year ago and nobody ever ordered anymore), so we just used another, even older, CCR2 antibody we found stashed at the back of the freezer, just to hope for a possible miracle it might work.
Well, considering the circumstances, we didn’t really have high hopes for any of these antibodies, but we went through the motions, spent Thursday and Friday doing IHC with the 8 pieces of tissue (one PBS control and one nitrogen mustard per marker). When it got time to actually stain them under the microscope on Friday, we were supremely unconfident with the possibility of it staining. We waited a while, waited for it to stain, and I swear, it turned brown! It did. But I don’t know what happened because after we came back to the slides after lunch (when the mounting solution we used to coverslip them had dried a little) there was literally no stain to be found. None. No brown. Anywhere. Not a single portion of the cell had stained, for any of the antibodies. What a disappointment. But I guess we were also sort of expecting it because of the terrible antibodies. But it was still so sad to see nothing stained. And I keep having this insidious feeling that we should have just left it in the stain for longer, but I don’t think that would have made any difference. The worst part is you can’t really expect everything to be stained. Since the markers are so specific, you never know whether the slide didn’t stain or whether there’s just none of that cell type present. Regardless, we’ll see if we can take a picture of one of the more promising slides and get anything from that. We also ordered newer antibodies, which will come by next week, and we’ll try troubleshooting those antibodies to get some actual results.
But along with all those failures of IHC, we did another round of protein assays on Friday, where guess what! Our standard curve was completely whack (it had an R2 value of like .8) so we have to redo all of that as well! So many failures this week. We might as well have done nothing. But it’s okay, it was a learning experience. Not everything goes well all the time, adversity is expected. You just gotta live with it and move on.
There was one light at the end of the tunnel though. The wonderful Wheater’s Functional Histology Text and Color Atlas. Yes, it is a textbook, and yes, it’s meant to be for grad students, but oh my god I love ittttt. So much. Alexa brought it in for us to look at if we wanted to before scoring the slides, so I decided to look over the pulmonary section and I was hooked. It’s literally just a textbook going through all the possible tissues divided up by organ system and goes through cells and staining types and everything and I love it. It’s incredibly interesting and I couldn’t stop reading it, so Alexa let me borrow for a few days. Right now, I’m about 4 chapters in (after reading pulmonary and immunology - which is applicable to my research right now - I read both nervous tissue and system chapters, of course) and I really, really want my own copy. I also want the Pathology version, which goes through everything that can possibly go wrong, but that’s really expensive and is an actual med school textbook so I dunno if I want to go there yet. My plan is to read as much as I can from Alexa’s textbook, buy my own copy later, and actually learn the information instead of just reading it passively. That’s how much I love it. And I totally didn’t expect to like it this much, but I love microscopes and looking through them and now apparently histology.
But that was it for my week guys, it was pretty short and unexciting and not the most successful. But I learned a lot. The next few weeks will be a lot more IHC and protein assays and scanning slides and (ugh) counting cells as we try to get together enough data for a paper and start putting our poster together. Sigh. I can feel it coming to an end, and I don’t want it to. At this point, I’m seriously considering going into research and getting my PhD. Let’s see. Let my chips fall where they may, I’ll go where the wind takes me.
And as always, thanks for reading!
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