Reign Icasiano, George Lab UCSD, Week 2

Hello! Welcome back to my weekly updates from Team San Diego! This week at UCSD has been a short but fun one! Although I was only at work for three days, those three days were packed with a bunch of learning, new concepts, meeting new people, and of course, more rats.

Mon. June 16, Day 8-

Today was a longer day in the lab than I’ve experienced thus far. I started and ended my day in the wet lab at

A bit of our setup for IHC!

Skaggs doing immunohistochemistry (IHC) with Sonja. IHC is a lab procedure used to find and identify and visualize certain proteins in tissue samples. The whole IHC protocol or procedure, looked like this:

1. Prepare the brain slices
2. Blocking nonspecific binding and permeabilizing the cell membranes
3. Primary Antibody incubation
4. Wash
5. Secondary Antibody Incubation
6. Wash
7. Coverslip

You might’ve read, in my last blog, that I learned to plate the brains and coverslip them. IHC is kind of like an extended version of that procedure, adding in more steps like the washing and the antibody incubation. The purpose of our IHC was to validate that the virus we injected, was in fact there within the CRF-cre rat brains. CRF-cre is a rat strain where crf+ cells are a neuronal subtype, where cre is only expressed in those crf+ cells. The virus is dependent on cre-, so it should only express itself in the crf+ neurons. Even though there are only 7 steps in our procedure, it took a whopping 8 hours to complete it all in one day. The procedure in itself was quite simple in itself because it was repeating the steps just with different ingredients, however, it was very important that everything was done precisely and accurately to get the best results. There was a lot of waiting and downtime while the brain samples were being incubated in the antibodies, so during this time, I got to read up on a few papers, and Sonja taught me some concepts I had been confused about. I had also learned about what a “brainbow” is, and it’s a brain that’s stained into the different colors of the rainbow, hence, brain-bow! Although it was a long day in the lab, it was a great learning experience, and goes to show how meticulous and specific the ways of science are, and are only effective when done correctly.

Tues. June 17, Day 9-

Sonja and Elizabeth’s setup for flash freezing and brains

The biggest lesson I learned today was that collaboration in science, or just collaboration in general, is key to an effective outcome! Tuesday started off with Sonja showing me how to image the plates that had gone through IHC yesterday. It was really interesting to see what was going on in those tiny slices of the brain at the cellular level. When imaging, you’re able to manipulate what you want to see, and how you want to see. Through this, you’re able to decipher if what you did in your procedure was correct, or if you need to change a few things. As Sonja was going through the sections of each brain that was plated, she mentioned that the virus that they had injected had too high of a concentration and that there was too much of it, so the best solution to this problem would be to redo the injection, insert the virus in the same location with a smaller dosage and concentration. I’m very excited to hopefully soon be able to get to do some imaging myself! After our morning in Skaggs, we went to MTF to have our first round of dissections. I’m thankful that I had seen a rat be sacrificed on Friday, so I had much more confidence walking into the dissection room than I would’ve. During dissection days, most everyone I work with in the lab is there, and it kind of felt like a reunion! I had worked with all of these people separately but it was fun to see everyone all together. Our techs, Sarah and Joseph, were in charge of bringing the rats into the dissection room, as well as the post-dissection assignments, Ms. Molly was in charge of actually dissecting the rats as well as perfusing their brains, Elizabeth and Sonja were in charge of taking the brains out of their skulls and flash freezing them, and Max was in charge of setup and tubes! It was cool to be in a place where everyone had a role in what we were doing. Today, we only dissected 4 rats, two of which that needed to be perfused and two that had brains that needed to be flash frozen. Perfusion is where all the blood is taken out of the brain through a thorough washing protocol before the brain is taken out of the skull, and flash frozen brains are done after being taken out of the skull, with the brain being put in dry ice until it’s hard and makes a little “clink” sound. Although I’ve done

The plates from the practice brain I got to plate

a few dissections for school, these dissections were way different, and I didn’t even know that there were certain ways to even keep the organs that are needed! As mentioned earlier, Max, an undergrad student, was in charge of tubes. These were the tubes I worked so very hard on last week, writing which tubes belonged to which rat and organizing them neatly. The tubes had different colors to differentiate which organ went in which tube. From each rat, we took it’s blood, brain, spleen, and tail. Ms. Molly, when dissecting the rats, cut open the rat’s body to retrieve it’s blood and spleen, and also cut off it’s tail. After that, she cut the rat’s heads off and those heads went to Sonja and Elizabeth to dig out the brain from the skull. Once the brains were taken out, the perfused brains went into tubes filled with saline, while the flash frozen brains went in smaller cylindrical tubes. The blood and brain tubes were then given to Sarah and Joseph to take care of, respectively. After dissections, Sonja and I went back to the Skaggs wet lab, and during the rest of the afternoon I continued to shadow her imaging, read some more papers on IHC, and practiced plating more brains. I’m definitely starting to get more comfortable and more confident plating these brain slices, so much so that Sonja has tasked me to start plating important sections for her projects! This exciting mission would start tomorrow, and I was overjoyed to get the chance to work directly for Sonja’s research.

Wed. June 18, Day 10-

Ms. Molly’s side of the dissection room where the perfusions happen

Wednesday morning started off bright and early with a new group of dissections. In the morning, I met Max and Ms. Molly in MTF, where we were immediately put to work, getting the rats ready and setting up the dissection room! All of the dissections we were doing today were solely perfusions, so no dry ice was needed. Soon enough, everyone working the dissections soon came in to help and we got started. Perfusion dissections have a lot of downtime, so while we waited for the saline and formaldehyde to run through the rats to get all the blood out, we got to jam out to Single Ladies by Beyonce (AKA 2000’s music), making the most of our time doing nothing. Though, I did feel a bit conflicted having fun, dancing and singing with the music playing in the background while the rats were cut open under the fumehood…However, the rats soon joined in, boogieing to Beyonce because of their amazing music taste! All jokes aside, the rats didn’t really come back to life. Formaldehyde makes the rats tense up and makes their limbs and tails twitch. My mentors in the lab call it “dancing” which I honestly saw because those rats had killer moves and I definitely felt threatened as a dancer myself! Dissections took up most of the morning, so after cleanup, Sonja and I went to Skaggs to do complete a few missions before heading to the data analysis meeting later that afternoon. During this time at Skaggs, while Sonja continued to image, I started on the task she gave me yesterday, plating important sections for one of her many projects. I really wanted to do well and not screw these plates up, so unfortunately I only got done with one section out of the brain out of the six I needed to complete. However, Sonja said I could work on the plates later, and it was alright that I didn’t finish all of them right now. Soon after, we headed back to MTF to be a part of the data analysis meeting. In this meeting, we saw Elizabeth, Dr. Olivier George, and Liselot, the head of data and coding in the George Lab. This meeting was to talk about the most recent cohort of rats, the rats we dissected today, and how well they did for the GWAS (genome wide association study). I didn’t realize how hard it was to interpret the data that labs collect, and that some of the data points that get collected aren’t even used. Of course, this lab has been running for a while, so most of the rats did what we wanted or expected them to do, but since these rats so closely related to the human population, there were some outliers that were unpredictable. Sometimes these outliers got a “pass”, but a lot of them were excluded from the overall data points. There were many rules, like in coding, that went in to picking and choosing which rats passed and which rats were excluded, and these rules were curated by the members of the George Lab. In the three hour meeting, I got a good understanding of all the graphs, seeing how they worked, and what they were showing us. This meeting was the last thing I needed to check off for today, so once Sonja and Elizabeth were finishing up analyzing the final graphs, I continued to shadow them until they completed it!

Thurs. June 19, Day 11-

Shiloh, Azari, and I got matching henna at the farmer’s market!

Today was Juneteenth, so we got the beautiful day off! With the other Pinterns, we decided to for an outing in San Diego, first of course, hitting the beach, and then heading to a farmer’s market close by. That same farmer’s market also happened to have a cool bookstore nearby, so of course we had to explore that as well! It was very fun being with everyone, and especially being in the car hearing about the adventures of Rhianna and Bellamy, Azari and Shiloh’s cell cultures they’re growing in their lab at Scripps!

Fri. June 20, Day 12-

Today, I got another day off because quite literally no one was in the lab. So although I didn’t get the chance to be in the lab today, I benefitted the rest of our housemates by doing a little cleaning at our house. Other than that, though, I didn’t do much, and instead waited for them to come home. I’m so grateful to be put with the girls I’m with, as they have truly made this whole experience so memorable. Memorable enough to even have an impromptu karaoke night where many songs were sung, but my voice was definitely gone from having so much fun!

Sat. June 21, Day 13-

I’ve gotten to spend my third day of my four day weekend back in the San Diego sun! Today, the Pinterns and I took a trip downtown, where we got to explore the Little Italy Farmer’s Market! I now understand why this farmer’s market is so popular, everywhere you turn you’re tempted by each booth and what they were offering. Although I did purchase a few impulse buys, I also got my fair share of delectable free samples. Azari and I shared a delicious strawberry and Nutella crepe as well as a coconut sundae topped with mangoes and sticky rice with Lana. Clearly, I had an amazing time at the farmer’s market. After Little Italy, we went back to the area we were at on Thursday because we couldn’t get enough of the bookstore we found. Luckily for us, a vintage clothing store was set up near the store, so we got to browse in some clothing too, which was really fun. Soon after, we headed home to rest up, which officially concluded our second week in the lab!

Fun photo op while we were out!

Sun. June 22, Day 14-

Sunset on the beach!

It wouldn’t be a San Diego Sunday with our little Pinhead group if we didn’t head to the beach. I was surprised we didn’t go yesterday, as we’ve been hitting the beach every chance we got! In the morning, Lana, Maiya, and I went to the gym to get a workout in, and truth be told, I was definitely sweating. So as a little treat, we went to a coffee shop near by to indulge in a nice reward. When we got home, Azari and Shiloh were up, so we cleaned the house and rested a bit before heading to Del Mar for their beautiful beach. Our goal as interns is to never be at the same beach twice, and so far, we’ve done so good! Del Mar was absolutely breathtaking, and it was surreal to be watching the sunset while the waves crashed on the sand. The waves at Del Mar were a bit intense, they kind of attacked us even though we did nothing to them! However, we had so much fun and many pictures and videos were taken!