Hello, my name is Ian Snapp, and this is the fifth week of my neuroscience internship at the Scripps DNC. To recap, I am working under the Ye lab, studying metabolism within the nervous system. This research requires a focus on an incredibly minute scale, involving molecular biology and biochemistry manipulating DNA to change metabolic patterns inside of neurons.

Things I Have Learned

To add to the lab procedures, I learned about IHC, staining, and AAV titer. To start with, IHC means immunohistochemistry and it is used in order to locate proteins called antigens and potentially bind antibodies to them. In this case, a primary antibody solution suspends brain slices (produced last week) and binds to the antigen of interest. Once these antibodies have been located and bound to the antigens, a secondary antibody binds to the primary antibody so tags can then be attached. The tags or tracers are chemicals that are dyes or fluorescent to highlight the target proteins produced by sample DNA. AAV titer, on the other hand, is just binding the capsid proteins (shell of a virus) to a plate, shining light at it, and examining the amount of light that was absorbed from the specific wavelength that the protein typically absorbs. This, in conjunction with a standard curve to form a baseline comparison, allows for a relatively precise calculation of the AAV concentration in the solution.

Monday

As per schedule, the week started with a lab meeting lasting until around 10:30. Once the lunch break was finished, I centrifuged the bacterial solutions into a pellet (the particulate matter/bacteria that was suspended in a solution) and froze it at -20 degrees Celsius because we were out of stock on the filters needed to do a midiprep to isolate the DNA. Once I finished this quick procedure, I watched as Dawn performed an IHC with the primary antibody on the brain slices from last week.

This is the workbench as Dawn was binding the primary antibodies.

Tuesday

Tuesday started with Dawn performing another IHC with the secondary antibody. Briefly, while she was working on that, I picked colonies from more bacteria samples containing sample DNA and transferred them into an LB broth solution to incubate. To bring the day to a finish, Layao and I resuspended the HEK cell-pellet containing AAV. Once the HEK cells were resuspended, we lysed the solution, dissolving the cells and releasing the AAV. We did the same to the supernatant solution the HEK cells were suspended in previously. After the AAV was released, the solutions were centrifuged down, the virus-rich solution was removed, placed into a separate tube, and frozen overnight.

These are the LB broth solutions that I suspended the bacteria in.

Wednesday

Wednesday started with the simple tasks of spinning down the bacteria incubated on Tuesday and isolating the AAV virus. Unfortunately, the midiprep filter columns hadn’t arrived yet, so the bacteria pellet would need to be frozen like the other two on Monday, but there were no centrifuge tubes clean, and most of the other glassware was dirty. So it quickly turned into running two loads of dishes, three autoclave cycles (including a TC and LB broth restock), and, while I was at it, cleaning up the unkempt sink station. All this cleaning lasted until around 6:30, but I was able to catch a bit of the final AAV purification in the afternoon. The purification was essentially layering four solutions with different, known densities into a centrifuge tube. Once the solutions were added, the tubes were centrifuged for a couple of hours, pulling the AAV into the third layer of the solutions (due to its density aligning with that layer in particular). This was then extracted and filtered. (I believe this is where the purification ends, but I may have missed another step due to the autoclave cycles finishing up around this time).

These are the centrifuge tubes filled with the solutions used to isolate the AAV.

Thursday

Thursday had a late start, really only beginning after the 1:00 pm group meeting with our PI Li Ye. Once the meeting had finished, Dawn started the primary antibody staining of the brain slices and Leyao started an AAV Titration. I went back and forth watching both of the procedures, but it was essentially watching Dawn try to suspend and coat thin brain slices with a solution then leaving it on a shaking plate (used to agitate the solution). Leyao was using a known standard, curve protein solution to set a standard curve and then applying different concentrations of the viral solution to the plate to test it with a machine that shines light at it. To finish the day, Leyao started a primary cell culture, dissecting pups, suspending cortical neurons, and leaving them in a large multiwell plate, because all the other primary cell cultures became contaminated from bacteria.

Friday

Friday was spent mostly watching Dawn perform staining on another batch of brain slices (multiple different proteins to bind and stain within the brain) but I briefly watched as Leyao replaced the media in the primary culture. After those were finished, the day was essentially over and everyone went home.

Saturday

Saturday wasn’t the most eventful day. I spent most of it working on SAT practice, planning college lists/admissions, and catching up on some missed sleep. In the afternoon, I drafted the blog, then went on a walk around the local area, then came back and straightened up the house. There wasn’t really much other than that.

As a side note, most of my week was spent doing SAT practice and planning out university details because the work week was relatively light compared to most. I was just clarifying to say that most of the week was quite sparse compared to what may have been conveyed here.

Thanks for reading again, only one more week to go.