This week was the week of learning. Along with continuing to do the procedures that we had already learned, we learned so so so many new things. Most importantly, we finalized what we’ll be doing for our final “project” that we will eventually present in August during the Liberty Science Center Symposium. This week, because it was another THED week, was also spent working with a lot of other grad, undergrad, and high school students (including one of the smartest people I have ever met). And in case you haven’t read my earlier updates, here’s the first one.
This week started off with us being introduced to literally the smartest person ever. I’m not gonna say his name, but let’s call him James. (He is actually the aforementioned high school senior/college freshman). His dad is the PI of a lab that works really closely with my PI, so there’s generally a good relationship between our labs. But anyway, he is a genius, incredibly smart, and is going to publish a paper at the age of 18!!!! Like an actual paper. Not one of those where high school friends brag about writing a random abstract and getting it looked at. It’s one where he spent 2 years doing all the independent research (with Alexa’s funding cause he is technically not employed by the lab) and found actual results that have global implications. He was actually an LSC student too, but that was more of a formality; he’s been working in his dad’s lab since he was 8. (No joke.) Now, it’s a little hard to separate his supposed genius from the numerous opportunities he’s gotten by having his dad as a PI in a distinguished lab, but regardless, he is incredibly smart. And he’s so chill too. (Except he’s an Eagles fan. Why is no one else in this entire lab a Pats fan?!) Well, that’s enough about him.
Anyway, James taught us how to count the cells that I had Giemsa stained - we were looking for neutrophils, monocytes, macrophages, and foamy macrophages (which is what his research is focused on). That took a ridiculous amount of time, because we had to count 1000-2000 cells a slide, and I don’t even remember how many slides there were. After that, he showed us how to run a Native Western blot, which is the more complicated cousin of the normal, reducing Western. We pretty much hung out with him the entire day, just talking, not really doing much in terms of science. (Except for that tortuous cell counting - for those of you who don’t know how it works - you literally look at the slides under a microscope, and going frame by frame, try to distinguish the incredibly minute differences between cell types, counting maybe 15 at a time, and literally punching a lever - like a typewriter - to count that cell. For a thousand cells. The entire slide has 100,000 cells. But we just extrapolate the numbers, thank god.)
Tuesday and Wednesday were pretty chill days (we got Moe’s for free on Wednesday!). We ran another IHC, this time with iNOS antibodies. This was a fresh batch of antibodies, so before we can start running them on multiple slides for actual data, we have to find the optimal antibody concentration, so the slide doesn’t over or understain. Thankfully, a post-doc at UPenn (who did his PhD from Rutgers and is still close with Alexa) had ordered the same batch and had already done some IHC with it, and told us to start at around a 1:200 concentration, that is, 1 ul of antibody per 200 ul of PBST/NGS (goat serum in a saline solution). So we just thought what the heck, we’ll start with that. And like magic, it worked like a charm. Mostly. The affinity was a little less than previous antibodies like COX2 and HO-1, so maybe we’ll try it at a little higher concentration next time, but this works too. But we did that IHC over the two days, and on our off time helped another grad student in James’s lab with some miscellaneous things she had to get done, like making random buffers for flow cytometry and westerns and labeling eppendorfs and what not. On Wednesday we also learned how to do a BCA protein assay, which is an incredibly easy but time consuming procedure. It’s literally just putting samples and a working reagent into a 96 well plate, letting it incubate, and using a spectrophotometer to read the color change - more purple indicates more protein concentration. It doesn’t really require any thinking, but can give really interesting results, and so of course, because none of the grad students or other researchers want to do the boring stuff, we got saddled with like 50 samples of BAL and were tasked with doing a protein assay on each.
Thursday, we got to sit down with Alexa and actually talk about what our project was going to be. Originally, we going to do a part of her nitrogen mustard-gadolinium model, but she doesn’t know when the endpoint for that will be, or if our PI will let us actually use that. So our final project is going to be looking at the effect of the FXR (Farnesoid X Receptor) gene on the development of pulmonary fibrosis and inflammation. That was actually the first takedown we observed, so it feels right to follow the project from beginning to end, instead of joining in at a random point. The only problem with having the FXR project is that the nitrogen mustard control group (as in the group of mice who were injected with nitrogen mustard but didn’t have the FXR gene knocked out of them) all died before the takedown. So technically there’s nothing to compare the FXR -/- (who did have the gene knocked out) mice to. We’re trying to get lungs from other mice for the data, but it all depends on what the other projects are and if they’re working with nitrogen mustard and FXR too. Otherwise, we’ll just work with what data we have and make conclusions comparing it to the PBS (as in no nitrogen mustard) controls.
On Thursday, we also did a bunch more protein assays, listened to some more SURF presentations, and helped James with another Western (read: annoyed James with our terrible technique). This time, it was a normal Western, and didn’t have many consequences if it went wrong (because it was going to have to be done one more time anyway), so he didn’t feel bad about letting us do most of the work. So we did pretty much everything (with a lot of his help) and we only failed a little at putting the samples in the incredibly tiny wells of the gels. These were actually almost 1 year old samples that had to be dug out from the abyss of the storage freezer with (I’m not kidding) a hammer and screwdriver as a makeshift pickaxe because they were so frozen. The paper involving those samples was submitted about a year ago, but while it was in review, people said it would be that much better if they could get this one piece of data from this Western, so here we were, doing the Western. A Western blot is actually a 3 day procedure, but we only did the first day, because we were going to be busy on Friday and were not masochistic enough to come in on a Saturday.
On Friday, along with it being the last day of THED and therefore the last day of free food (noooooo!!!), we were supposed to work with a different grad student to help her score the FXR slides the entire day (there were a lot of slides). Scoring is basically where you are blind to which slides belonged to which control or treatment group, and you look at them under a microscope and give them a score from 1-8 (or something different, depending on the scale) depending on the level of whatever you were looking for - in our case, fibrosis. This grad student ended up calling in sick so we were left with literally nothing to do. Alexa was busy with THED, we didn’t want to start an IHC cause no one could come in on Saturday to finish it, we had exhausted samples to do protein assays on, and James was busy. We ended up replacing the bench paper in our lab (it was in such a bad state), reading papers about FXR, and eating with the THED students (where I was repping my blog - if any of you THED kids I talked to is reading this, comment!). In the afternoon, we were on our way up to another lab in the School of Pharm building (because the cooling had shut down in our labs and it was torturous to stay there) when we saw the undergrad who we had met during the atherosclerosis lecture (who happened to be my classmate’s sister) and she needed help looking at some slides so we thought, why not. Those slides actually ended up being the ones we were supposed to be scoring with the grad student (what a coincidence). But instead, cause none of us knew how to score it properly, we just looked at the slides and took pictures of the interesting areas where we saw a lot of fibrosis. Those slides and pictures will eventually end up being a part of our final paper/poster/presentation.
Well, that was my entire week. A little unorthodox, we literally just did whatever we could find, but we learned a lot. Plus we met a lot of awesome people. Plus I got to use the laparoscopic machine again (only once though) and got it under 7 minutes with a pretty good circle. (I didn’t figure out until the last time I tried it that I could rotate the trocars (the instruments that go inside the body) which made the entire thing so much easierrrrr. Smh. Wasted my time practically injuring my wrist rotating it to ridiculous angles.) Next week, we’ll start actually working on our final project presentations and maybe even analyzing some of the data we’ve collected so far.
Thanks for reading!