There are Good Days, and There are Bad Days for Science

Hello everyone! For this week’s blog entry I’ll bring you through a day in my life working in the Bilbo/ Eroglu lab. My day begins with an 8am wake-up and a half hour of frantic pre-work preparations. Somehow during this time I manage to shower, dress, make breakfast, pack lunch and run out the door to catch the 8:30am Swift Express bus to West Campus. This bus is crucial. Missing the Swift Express means being late for the 9am start of the work day. 

Once in lab, I check in quickly with my mentor, a final year PhD student, Carina, about my daily schedule. Because most of my time is spent on software/ equipment owned through Duke’s Core Research facility (expensive and/ or large equipment which the University only owns a few of and are thus shared between all of the labs on campus) I am able to dictate my own daily schedule based upon availability of the Core machines. It is through this University program that I am able to image slices of brain tissue on a Zeiss 880 Confocal (which is super cool!), deconvolude these images (reduce background noise and make more clear) on a software called Huygens, and create 3D reconstructions of the microglia I image in software called Imaris. 

By 10am I am set up to use one of these softwares – Zen (used on the confocal microscope), Huygens, and Imaris which takes up most of my days, depending on what stage in analysis I am in. While I am grateful that the University has licenses to these programs which allow me to visualize microglia cells and synapses within the brain, it is incredible how much of my day is spend troubleshooting such expensive software. 

By noon, Imaris has probably crashed at least twice (see attached photo), and Huygens failed to process my images. While I troubleshoot or re-do lost work, I eat lunch, usually snacking throughout the day as opposed to taking a long break. I take “lunch” time to organize data which I’ve acquired, schedule more time on Duke Core Research machines, or chat with lab members. 

3:30pm: Coffee time!

You can usually find me in lab until around 6pm, after which I’m either too hungry to go on without proper dinner, or too annoyed at how long a particular program is taking to render my data. I trek home, catching the Swift Express back.

In the evening I gym, cook dinner, and watch some Netflix until I crash for the night, ready to do it all over again tomorrow!

Sometimes if I wish hard enough, it responds

Me with the scope!

A Morning Blend of Thoughts

On most mornings, I’m lulled into a new day at lab by the hum of a Krueger machine spouting out warm, ripe coffee. As decisions run through my head and I reach for the hazelnut creamer cups, a flurry of brief philosophical conversations with my mentor flutters into my head: Does free will really exist? Are decisions really ours to make? Maybe in what I thought was a free will decision in choosing to put creamer in my coffee was merely the manifestation of an innate evolutionary urge for creamy food, a predetermined course of action I am just biologically hard-wired to. Like this, I feel like I am learning more than just techniques but different modes of thought everyday in the lab.

However, before any intellectual discourse with my mentor, I must tend to start any experiments that run on a rather rigid schedule. To set up the experiments for the day, I gear up with a full outfit of personal protective equipment and head into the animal room to retrieve mice. I then transfer them to a private suite, where I randomly place mice of all cages into separate enclosures on top of a perforated stand. For an hour or so, they are left to roam freely in their enclosures as they habituate to new scents and sights of the room. When it is my turn to test the sensitivity of these mice, I frantically evade their droppings and track their most subtle movements as they scurry around in their enclosure. Towards the end of the day, I will usually analyze and display the data I’ve collected on an Excel spreadsheet and run multiple statistical tests. Science is about robust data and multiple lines of evidence. And so, on select days I would complete immunohistochemistry to visually verify the behavior of certain cells of interest. To do so, the first step is to section different tissue using a cryostat, almost like a deli slicer. In the next step, I would stain for different types of cells by marking different cells with colorful tags using antibodies. When it comes time for these tissue to be imaged, I would get to operate a powerful imaging scope to take multi-layered photos of the samples. 

It is in between these experiments when my mentor would test my understanding of the fundamental science and then proceed to thoroughly explain the purpose and thought process behind each experiment. Currently, I am working on an experiment that involves siRNA, which knocks down IFN receptors in mice temporarily to eliminate any developmental issues as confounding factors that could accompany knockout models of mice. To help aid my understanding, my mentor doodles precisely illustrative diagrams to accompany each explanation. It is also during this time when I get to ask any questions on my mind or discuss any philosophical inquiries that somehow relate to our project. Day after day, lab feels like an intellectual playground, and I am grateful for the freedom my mentor allows for me to wonder. From the elaborate and clarifying explanations my mentor consistently provides, I am more than ever opened up to the intricate beauty and cleverness of scientific research. 

Take a Walk in My Lab Shoes

What’s up readers? This week I’ll be sharing a typical day’s routine from my time in the lab. My days start at 9 in the morning when I usually get to lab. I like to bike to work in the mornings from my apartment to wake myself up. My morning schedule completely depends on what experiments I ran the night before, but usually my mornings consist of completing Western Blots or harvesting RNA from my cells. Western Blots require an overnight antibody incubation step, so on days where I run Westerns, the next morning’s first step is to complete it with the necessary washing, secondary antibody incubation, and image development. On the mornings that I’m not developing blots, I often have RNA harvesting to do. One of my most common experiments is comparing RNA levels between different cell treatments using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). These experiments often require treating cells with certain drugs, some that inhibit our kinase of interest, others that inhibit other kinases, and others that act as control drugs. These incubations are often 24 hours, but can be as long as 72 hours depending on the drugs used. On days that I harvest RNA, I usually set up the RT-qPCR experiment. This involves an intermediate step known as the cDNA synthesis which converts the sample RNA into DNA for the PCR reaction using reverse transcriptase. After this I set up the RT-qPCR reaction. The entire procedure takes about half the day to set up, but it yields really good data. By the time I’ve finished developing my blots or setting up an RT-qPCR, it’s usually around lunch time. Most of the time I pack a lunch, but sometimes I’ll head to the Hospital Cafe or an off campus restaurant for lunch with some other members of the lab. If I eat in the lab, I like to analyze data or read papers during my lunch. I’ve found that there’s always more to read, and especially since my project is relatively open-ended, there are lots of papers out there that would aid my search for new mechanisms of Abl kinase mediated metastasis. After lunch is usually when I finish my tissue culture work for the day. I keep around 6 cell lines growing at all times, and to keep these cells healthy and happy, I have to dilute them for growing space and replace the media which they grow on every other day. Most of the lines I keep are lung cancer lines, but I also keep a line of 293T cells for the virus experiments that I occasionally do. In addition to regular upkeep of these cells, on most days I need to set up cell experiments. Often these are drug treatments, but they also include virus transductions, cell viability screens, and trans-well migration assays. For these other experiments, I have to count the cells that I have using a (very carcinogenic) dye, calculate the correct dilution ratios, plate a precise number of cells, and if I’m applying drugs, dilute the drugs to appropriate amounts. My tissue culture work can take as little as 30 minutes when I only need to split my cells, but it can take upwards of two hours on particularly busy days (usually fridays). After tissue culture, it’s often time to call the day quits. But if there’s extra time, I will usually run a Western Blot gel of do a quick cDNA synthesis to get a little ahead for the next day. I’ll usually bike home from work, relax for a little, cook dinner, and head to bed to rest up for the next day! My days can be pretty diverse depending on the experiments I need to complete. I love the variety of my schedule. Each day brings new challenges and new data to show to my lab-mates and to put on my poster. Although the 9 to 5 research life is long, I love my days spent in the lab. I’m nearing the end of my summer research, so in the coming weeks I’ll be finishing my research and preparing my poster. Can’t wait to check back in next week!

-Brennan

Lather, Rinse, Repeat

Building a gene regulatory network, I think, is sort of like solving a jigsaw puzzle. Little by little, discovering which pieces go where–through logic, through the process of elimination, through experimentation. My weeks in the McClay lab have usually looked a little something like that, each day a slow and steady step towards creating something that is bigger, more complete, and eventually can tell a story.

Analyzing the relationship between genes means having to carefully select and examine the behavior of each gene. The process by which we do that takes a week, or two at most, and so my time in the lab has been a pretty steady weekly routine of pipetting, incubating, and washing. Rather than a day in the lab, then, I’ll be briefly describing a week–four days that lead up to the final day, the final reveal.

The first step that I take is creating the probe of the gene we’re looking at, which will allow for us to locate the expression of the gene. This process normally takes two days, as it requires a good amount of time for incubation. The first day involves a lot of pipetting, in order to prep the plasmid for use, while the second is the actual synthesis of the plasmid. Both are rather short days–they take only about two hours or so, as well as some waiting. While I wait for the plasmid to incubate, I usually spend the time going to lunch and enjoying the sun.

In-situ hybridizations are next, and each lasts three days. After we have prepared the well plates with embryos of different stages, the process involves a lot of washing as we prep the embryos for antibody staining in order to witness the expression change over time. These are a very systematic two days, with short intervals of waiting time and repeated activity in order to ensure all undesirable qualities are washed away. These days are longer and always busy, and I still haven’t figured out the best time to eat lunch, but it’s interesting because there’s always something that needs to be done. The last day of in-situs are the most important, though, because that’s the day of staining–the day we’re able to obtain the gene expression data we’ve been prepping since the beginning of the week. The hardest task of the day is learning at what point the embryo is done staining, and when expression of the gene has reached its utmost point. My mentor still helps me with this, but once I think it’s done, I stop the reaction. Sometimes, the wait can last hours, but I check on the embryos every 15 minutes or so. However, like the previous two days, it’s a tricky and busy day.

Sometimes, days end short or go longer than expected. But I’m glad to have this kind of routine, an understanding of what to expect, and a kind of independence that allows me to do things at my own pace, on my own schedule, and to have the authority to dictate when I can go on my lunch breaks or not. I feel a lot more respect in that sense than I did in other working environments, and have really been enjoying the past few weeks working at the McClay Lab.

Honorable mention: last Monday was a special day, because instead of the usual, my lab and I went out to the marine lab in Beaufort to collect sea urchins! It was hot and the sun was beating down strong, but we had a great time out on the sea, feeling the breeze and taking in the views. I came away with a little bit of a sunburn, and the memories of an exciting adventure.

Still Haven’t Found the Elevator Yet

Every morning, I’ll walk into the LSRC and I’ll see stairs—three long, tall flights of stairs I have to walk up in order to get to lab. After making my way to the third floor, I’ll round the corner into the Adcock Lab, set down my things onto my desk, and catch up on the messages in the lab’s Slack page. My mentor Abby will often stop by and we’ll set up a time to meet that day. This meeting will usually regard problems that need trouble-shooting or tasks that need to be done that day, and we’ll discuss what kind of work I’ll need to do for the next steps of the experiment. The daily work I do differs based on the stage the project is at. We first began by creating the experiment itself, a computer-based task. My mentor developed and coded the first task of immediate curiosity on conditional engagement, and I prepared the second task, a memory test. Since the experiment is centered on videos of art and drawings, our memory test essentially consists of comparing screenshots of old videos with new ones—command-shift-4 has now become one of my best friends! I also compiled relevant questionnaires and inputted each question into the task. Since finalizing these parts, we’ve sent out our pilot studies and gathered our first data points—which brings me to the work I do today! 

Since we send out our study in batches, I’ll spend my time cleaning the data received from the latest cohort, such as removing data from participants who didn’t complete their tasks. Our experiment has two-parts, so it’s also important to confirm that the subjects in each pool match each other. I’ll cross-check subject IDs from part one with part two, allowing us to make sure that the subjects who completed both tasks receive their full compensation and eliminating those who have not. 

I’ll then do some preliminary data analysis—mostly just some basic data science manipulations using Python. I’ll first reformat the data sets to look more organized and show only relevant information. I’ll then usually try to map trends through graphical analysis, as well as examine individual outliers, errors, and uncertainties. This way, I can practice my techie skills but also still gain knowledge from the cohort’s data! This is one of my favorite parts of my work, as having more data points strengthens and clarifies trends and brings you one step closer to a completed puzzle. It’s also an incredible feeling to realize that your work is real—the data is tangible, concrete—real!! Some days are slower than others, but that’s research. Research is for discovery, and as long as my little efforts contribute in some way to that truth or that discovery—I’ll be happy. 

A Day of Neurons

A typical day for me in the Gong Lab consists of me helping my mentor, Emily, with her project on how visual manipulations in virtual reality affect the firing pattern of place cells in mice. This project has been ongoing for a couple of months, and the data collection of the mice running through different conditions in the virtual reality set-up has already been completed.

What I am doing now is selecting through a list of computer generated masks and determining which circled areas are neurons and which are just arbitrary areas of fluorescence. This is necessary because the algorithm that is used to circle the potential neurons is not always accurate and may outline a group of pixels that does not represent a singular neuron. For example, the outline might circle two neurons, the space between two neurons, only part of the neuron, part of a neighboring neuron, etc. What I have learned from this particular task is that science is a slow process and that while looking at neurons firing on a computer screen may seem like a repetitive and mundane job, it is just as important towards the final results as any other step in the research project. 

Additionally, Emily is starting to train new mice, so I am getting some more hands-on experience with the mice and with the wet lab aspect of research by helping with the water restriction process. This process consists of giving a small amount of water to each mouse only once a day and recording the mouse’s percent weight loss. This water restriction process is necessary because in the experimental and training period, the mice are given water as positive reinforcement to reward their behavior.

In my free time at the lab, I am also working on improving my MatLab skills by practicing with some problem sets. These problems focus on the areas of MatLab that are important for the coding of the video processing program that we are currently using to select through the potential neurons. Once I have mastered the MatLab skills in these specific areas, I will be able to better understand how the computer is generating the masks around the areas of fluorescence and hopefully be able to use this deeper understanding to make more accurate neuron selections.

Working 9 to 5!

Everything’s routine now. The moment I open the door to the Bryan Research Building, a quick rush of AC floods over me. I click the “^” button, step in, click the “4” button, step out. I round the corner, smile and wave to Grace in front of me, plop down at my desk, turn right to say hi to my mentor Jenny. I check the board, scanning the pinned schedule that Jenny and I write on the past Fridays. It will be a mixture of different procedures: burr hole injection surgeries, perfusions, brain slicing, mounting brain slices, imaging, and coding, respectively. Everything must be done in order, and each step takes time, leading to weeks worth of waiting to obtain data. 

Each procedure is intricate and cannot be rushed. 

I usually begin my week with the first procedure: burr hole injection surgeries. I carefully drill a hole into an anesthetized mouse’s skull in order to inject fluorescent tags called tdTomato. After a mouse receives a viral injection in its brain, it takes at least two weeks for the virus to be expressed, or visible through a microscope.

The snowball effect begins.

After two weeks, the mouse is ready to be perfused, and the mouse’s brain must sit in PFA overnight. After a night, the mouse’s brain is rinsed with PBS three separate times in 15 minute intervals. After approximately an hour, the mouse’s brain must be submerged in 30% sucrose for at least a day. After a couple days, the brain is be manually sliced into delicate, thin slices and placed in PBS once more. After the brain is divided, the brain slices are meticulously mounted onto glass slides where they must properly dry overnight. After a night, the researcher must make time to image every brain slice under a microscope connected to a camera. After taking pictures of the injected brain slices, the data must be analyzed in old or new MatLab code. 

After this long process to collect data from one mouse, something could have gone wrong at any stage. The injection may have been too deep or too far to the left, the brain may have been damaged during perfusion, the brain could have been sliced at the wrong angle, the injected area may have been physically stretched out during mounting, and more. Science is slow, and I never understood why researchers say this so often until now. 

Despite the weeks it takes to collect data from one mouse, I’ve learned to appreciate all researchers who have brilliant ideas and work day and night to generate data that may or may not significant. I take Dr. Glickfeld’s words to heart: “We don’t ever hope to see a certain result.” Anything that comes out of experiments will contribute to science in some way. 

I have a rhythm at work now where I can easily come into lab to get into a flow. I’m sad to think that BSURF only has three more weeks left, but I hope to hop back unto the Glickfeld Lab once the school year starts. I love the work here, and even though I don’t have a lot of neurobiology background, I am happy to learn something new everyday. I can’t wait to see what these next three weeks will bring! 

Ma Vie Quotidienne

Bonjour tout le monde, this week I’m here to detail what a typical day in the Yin lab looks like for me.

Each day, my schedule heavily depends on the availability of the operant conditioning boxes in the lab. Recently, I’ve been scheduled to use them in the afternoon, which means that I’ve been splitting my day into two segments⁠— in the morning, I’ll focus on analyzing the previous days’ data and graphing it, and in the afternoon, I’ll work on gathering more data.

To analyze and graph my data, I take a file from a program that tracks my mice’s behavior called MedPC and run it through a Matlab script that interprets the numeric codes along with each timestamp for when the mice press a lever, put their heads in a window to get a food pellet, or receive a reward. Matlab spits out a new excel spreadsheet and I then use programs like Excel, Graphpad, and NeuroExplorer to visualize the dataset and compare how mice act during stimulation and during nonstimulation periods. Around 12:30 or 1, I’ll hear a grumble in my stomach and head to the breakroom or a nearby eaterie to chow down the lunch I’ve packed for the day.

After satiating my hunger, I come back to the lab and enter the mouse house. It’s here that I weigh the mice and determine how much they should be fed for the day. I then put my mice in the operant learning chambers, attach optogenetic fibers and set up the stimulation program if necessary, and let them get to work. After they’re finished, I’ll put them back in their cages, clean the boxes, feed the mice, and tie up any loose ends on any data analysis from the morning.

I love that as the weeks have gone on, I’ve become increasingly independent in the lab. I still meet with my mentor, Francesco, two or three times a week to go over my results and work through any new ideas, but most of the day, I’m left to my own devices. It’s highly reassuring that Dr. Yin and Francesco have placed this type of trust in me, and now that my results are starting to come in, I hope they are proud of the work I have done. This summer in the lab has been a dream— often working until 6 or 7 at night and coming in on the weekends to do experiments seemed daunting at first, but now I understand that if you are genuinely interested in the work you’re doing, it’s much less taxing even if the time expenditure is much greater. I am thoroughly enthused to present my work soon to my mentors and peers, and I’m happy that I have been placed in a lab that trusts me and continues to keep me engaged and motivated.

All in a Day’s Work

“Choose a job you love, and you will never have to work a day in your life.” ~Confucious

I truly love the work that I do and the people I work with. I am so lucky that my days are not only filled with cells and amazing science, but laughter and heartfelt conversations as well.

As far as the science goes, cells and protein take up much of my time. Every other morning I need to split cells and change their media so they stay alive and happy. Often, after I split my cells I plate them so that I can do treatments on them the next day. The most important part of cell culture is aseptic technique. I have to make sure that I do not contaminate my cells in any way. Since I have two different lines, it is very important that I keep them separate.

Another very important part of my day that is often overlooked is the math. I have to make sure that I plate the right amount of cells and media so that both of my lines are at the same confluency to ensure that the most variables are controlled for. I do this by counting the cells and making sure to plate an equal number in each well. I also have to use math to make sure I have an equal amount of protein in each gel well and to make sure I use the correct amount of antibodies.

Throughout my day I follow many different protocols and use a lot of different equipment. I have learned how to do DC protein assays, to do Western blots, to collect protein, to do treatments, and to analyze my gels. When I am not working, my lab is always talking and laughing together. We have plans to go to baseball games, make dinner, and to go to a Young the Giant concert. Some of us have even been on 8 hour car rides back home! I have had a great time in my lab, both with the science and my coworkers.

Rodent Routine

Each morning, I start my day by becoming an astronaut.

Or at least that’s what I look like after donning the extensive PPE required to enter Bryan Research’s basement mouse colony. I spend about 3 minutes donning a blue full body suit, shoe covers, a hairnet, a face mask, and gloves, for a trip into the mouse room that takes me 30 seconds. Nice. But it’s all so we don’t give the mice our nasty human diseases, which is pretty important. After collecting my six male mice (Fatboy Slim, Tarzan, Pavarotti, King Arthur, Charlie Chaplin, and Big Chunkus, aptly named based on their character), I head back upstairs to the Mooney lab to begin their experimental sessions.

For each of my 12 sessions (6 morning, 6 afternoon), I fill the chamber with either air or heliox and put one of my females in. After placing one of the males in the chamber, I start the webcam and the Spike2 microphone software to record their vocalizations and begin recording their mating behaviors every 10 seconds. When the session is over, I clean the chamber, change out the mice and hope I don’t get bitten, and begin the next session. It’s interesting, some mice are more sexually aggressive and have high proportions of rear sniffing and mounting, whereas some are more like nice guys who prefer to groom the female. Some are great vocalizers (eg. Pavarotti), and some are pretty silent (thanks Charlie Chaplin). All of their individual differences are eventually averaged by my trusty Matlab programs to create exciting sheets of data.

To turn sound files and Excel spreadsheets about behavior into usable data detailing mean pitch per behavior, amplitude per behavior, rate of vocalizing per behavior, and so much more, I start by importing the sound files. A 20 minute sound file takes about 45-50 minutes to convert into a Matlab file, so I import all 12 of the files into Matlab overnight. The next day, I’m able to find out the background noise of the files with the help of another Matlab program that generates spectograms, images of the sound file over a period of 10 seconds. I have to manually select periods of the spectogram where there’s no noise happening except the gentle hum of the air or heliox pumping in. This background noise plus the behavioral data I import gets reviewed by my final program that analyzes the vocalizations in the context of each behavior, subtracts out the background noise, and churns out all of the data I’ll need. After astronauticizing myself once more to return my tired males to the mouse colony, I set up the sound files for importing and breathe a sigh of relief.

That’s a typical day at the lab for me, although I’ve definitely had some bumps in the road. Once, I forgot to close Big Chunkus’ cage all the way after his session was finished. I went to retrieve him for his next session and saw he wasn’t in his cage. After panicking for a good 30 seconds and having the sudden realization that research buildings are probably crawling with lost mice, I opened his cage, only to find him clinging to the ceiling, munching on a pellet of food. I’ve also accidentally left my cage of females wide open, a field day for my particular batch of athletic and adventurous ladies who took the liberty of hopping out and exploring the desk around them. But I’m learning each day and can’t wait to see the final fruits of all this data analysis at the end of the summer and share it with you guys!

What to Expect When You’re Expecting

So this week’s blog post is centered around my day to day experiences in the lab! I’ve had the title for this blog post since the day 1 meeting when Dr. Grunwald suggest we use interesting, creative titles for our blog posts. Anyways, on to explaining what I do in lab most days.

Usually, I start my days in lab around 10AM after the morning session with either faculty in the Biological Sciences or an informational session given by Dr. Grunwald or Anna.

The first thing I do when I get to lab is check on my morning fly check. This consists of me getting my fly babies (if you cannot tell I am very fond of my flies) that are in the incubator out and examining the vials looking for empty pupae cases. We keep flies in the incubator (25 degrees celsius) because it helps slightly speed up the development of the flies without causing any disruptions to development or manipulating any temperature sensitive mutations.

After checking on my crosses, I either transfer the adults from a cross into a fresh vial with new food to expand the cross or I will look for male and virgin female flies in order to set up a new cross. To look for flies, I first have to turn on LED light on the microscope and the CO2 gas which is filtered through water and to this small nozzle (I can control the release of gas through this nozzle manually). I can then shoot CO2 gas into the fly vial (I hold the vial upside down during this process –it keeps the flies from drowning in their food) which puts them to sleep. I then remove the cotton bung which plugs the vial and slightly tap the vial to get all the sleeping flies onto a pad. This pad (I like to think of it like a bed) is made of a porous material and is also attached to the hydrated CO2 causing the CO2 to be pumped onto the surface of the pad keeping the flies asleep while I sort through them using a paintbrush under the microscope. After I collect the flies I need to set up a cross, the other flies get funneled in the fly morgue (RIP fly babies).

Additionally during this check, I will also transfer freshly eclosed flies into a new vial if I am building a stock (flies that are all isogenic) in order to expand the stock (as my PI says, it’s always good to have a back up). After I am finished checking on my crosses/setting up new crosses, I return the flies back to the incubator so that they can continue aging. I repeat these same steps in the afternoon before I leave for the day as a part of my afternoon fly check.

After my morning fly check, I usually have a few options as to how to spend my day until I perform my afternoon fly check. The first week fo BSURF, I spent this time reading different fly protocols and literature about Drosophila. I also took time to understanding more about the purpose of the crosses I will be setting up and the different phenotypes that will result from a particular cross. My PI also uses this time to teach me a new technique that I will need for something I will be doing later in the day. There have also been points during BSURF where I set up blocks to collect eggs from specific lines of flies to mount on slides which would usually take me about 2 hours depending on how many block positions I had filled.

Currently, I spend the time between my fly checks either working on my blog post (which I am currently doing), making mockups of my presentation poster, or thinking/ researching about the next steps in my experiment given the questions my PI brings up for me to think about.

In the afternoon, I perform my fly check mainly to see if there are any freshly eclosed females  of a particular genotype that I need to perform a cross. This is mainly because there is a window of about 8 hours that female fruit flies need to become sexually mature. Before this time frame passes, the female is too soft for a male to mate with thus the female flies I collect during this time have not mated. It is important for me to mate  female virgins because that ensures that I know the possible genetic and phenotypic outcomes of the eggs she will lay since. If I pick an older female, I won’t know for certain the genetic combination of the eggs she lays.

Here’s a picture of the microscope and the CO2 bed

A Typical Day

What I do in the lab depends on the day, but all of my activities revolve around glass slides. Some days, I have to do my least favorite task: polymerizing the glass slides. The procedure isn’t particularly difficult or unpleasant, it is just time-consuming. The upside is that once I polymerize two batches of slides, I don’t have to do it again for awhile. 

Other days are all about printing the antibodies on the slides. In the morning, I will take some polymerized slides to the Shared Materials Instrumentation Facility (SMIF) cleanroom to use the microarray printer. I have to wear a full body suit over my clothes, complete with a head cover, shoe covers, and a surgical mask. The SMIF printer is really precise and can print tiny spots of capture antibody. I print 24 assays onto each glass slide. Then I take my printed slides back to our lab to use the Biodot printer. The Biodot is also a non-contact inkjet printer, but we can use it to print trehalose pads and detection antibody as larger dots surrounding the capture antibody spots. Then I leave the slides in a vacuum dessication chamber overnight. 

The next day, I will test the assays that I printed. This means spiking a liquid, usually a buffer or fetal bovine serum, with different concentrations of the protein the assay is meant to detect. These serially diluted solutions are pipetted onto the slides and incubated for about one hour. Then I put the slides in a wash buffer, centrifuge them dry, and analyze them with our lab scanner. The computer program we use scans and quantifies the fluorescence on the assays. All of this data is collected, sorted in Excel, and able to be made into a dose response curve. The dose response curve is the big make or break moment. What you hope to see is that as the analyte concentration increases, so does the fluorescence intensity on the assay. If the dose response curve looks weird or doesn’t have the expected limit of detection, I have to think about what went wrong and try again. 

My days usually follow one of those three basic patterns. Of course, each day is still a little different. Sometimes there are lab meetings, free food leftover from other events, or lunches with friends. Most days the grad students bring their dogs to our office space, so I get to play with them whenever I have a free moment. The people I work with are friendly and patiently answer my questions about where things are and what I’m supposed to do. My only complaint is that because we work with delicate antibodies printed onto glass slides, I live in constant fear of dropping slides (or God forbid, a whole box of 30 slides!) or smudging the antibody spots. It keeps me on my toes.

If one more rat poops on me I swear to God…

A day in the lab is what you make of it. The quicker you are, the sooner you are finished with work. In my lab, it seems as though there is a hundred different studies being done at once. I have only one study I work on and it’s my job to complete it to the best of my abilities.

As soon as a arrive at the lab, I immediately get started. My working partner Graham will tell me what ha has already done and what I need to do. Without hesitation I get to work knowing that the more time efficient I am, the quicker we are done (something Graham is constantly reminding me of). I usually start with flushing the chambers and getting them ready by installing the syringe of Remifentanil. The dose of Remifentanil is based on the weight of the rat; therefore, we must weigh and prepare the rats before this step takes place. Weighing the rats is difficult as they wont stop moving. This makes it hard for the scale to get an accurate measurement. Once this is done the rat’s catheter is flushed to make sure blood clots are removed before the Remifentanil self-administration trial begins. A sheet is provided to see which rats get injections and with what they will be injected with. The trial cannot start until these rats have their injections making this step critical in terms of time efficiency. Graham has noticed that I have become very good at this and thus I am in charge of injections most of the time. Older rats are the best because they take the injections like a champ. The younger rats however seem to freak out and do their best to poop on you as much as possible. It is hard to treat an animal with respect when it does this but of course you have to. Once the rats are in the chamber, we start their trial via a computer. During the hour we wait, I usually feed the other rats while Graham cleans old cages. An hour later we must take all the rats out of the cage and flush them to prevent catheter clogging overnight. While I am flushing the rats, Graham is flushing the chambers and imputing data. We then put the rats away and make sure all the drugs are properly stored. We then get things on order for the next day and then head home.

Fly With Me

A day in the life of me in the lab is like a 90s sitcom, where you can predict what’s going to happen but each episode brings it’s own laughs. Each day in the lab is essentially a step in my experiment process, with it’s own victories and defeats, which keeps me looking forward to what each day brings.

First things first, I get to lab and check on my fly babies. I have around 20 vials of flies to take care of so I go in and make sure they’re alive and well. If it’s getting too crowded or if they need new food I flip the flies into new vials. I then collect whatever line of flies I need for dissection and take them to the dissection scopes. Dissection has come to be my favorite part of the experiments I do. After a lot of practice, I think I’ve gotten to a place where fly brain dissection has become far less stressful and actually enjoyable. I grab my forceps and the buffer solution and dissect the brains I need to, at a rate of about 15 flies per hour. I have to dissect a line in about an hour to prevent the brain tissue from deteriorating. I then fix the brains in a PFA solution and wash them thoroughly.

The next step in the process is antibody staining to target the DIP-alpha expression in the brains. We usually put the brains in primary antibody overnight in a 4 degree C fridge, so if this is where I am in the process on a given day, I will come in and get the brains from the fridge and wash them thoroughly to remove the primary antibody. After that I’ll put them in secondary antibody, which will ultimately give us the fluorescence we need to identify DIP-alpha expression under the microscope. This staining process occurs for 2 hours at room temperature and is followed by yet another cycle of washing.

Finally, all the dissection and staining and washing leads up to the defining part of the experiment: imaging. This is where we can see if any of our work paid off. This part involves mounting the brains neatly on a slide and using a confocal microscope to view the brains and any specific signals from the antibody staining. I take pictures of the data and use the results to determine which flies I need to dissect next, what antibody I should change, and start the process all over again.

FISH out of water (and into the lab)

At this stage of my project, I am staining brain sections with Fluorescence In Situ Hybridization (FISH) and then imaging them. The FISH procedure to stain the mRNA of interest requires one overnight incubation in order for the probes to bind to the mRNA. First, I block and wash the sections. I use salt solutions and buffers to poke holes in the membranes of the cells allowing the probes to enter. I pipette the probes onto the sections with a hybridization buffer and then allow the slides to incubate. The next day I wash the samples and either fix them or proceed with antibody staining. 

The next step is imaging the cells. I have signed up to use the microscope for a few hours each day in order to take pictures of the astrocytes. So far I have had difficulties with visualizing the mRNA. One problem is that the neuron specific antibodies block clear visualization of the mRNA. If anyone has any thoughts on how to troubleshoot this problem, let me know. To mitigate the second problem which is unspecific binding of the probes, I performed FISH without using the probes so we could have a control slide. I am working on using this control to be sure that the fluorescent spots I am seeing are really mRNA. 

When I am not working on my project, I work on various things for my mentor. Shoutout to Bel my amazing mentor for the summer. She has taught me procedures unrelated to my project from bacterial miniprep to co-culturing astrocytes and neurons. She makes sure I understand how the buffers and reagents work, as well as the overall biological systems the lab is studying. She has also worked with me on every step of my project, from perfusing the mice to harvesting and sectioning their brains to what I am working on now.

A day in the life

Dear Diary,

Today was a pretty typical day in lab.  I completed a phosphorylation assay using 10% DMSO to make sure that the assay works with DMSO.

I started out the day by stopping the dephosphorylation reaction that ran overnight.  The reaction mixture included dephosphorylated HipA, MnCl2, lambda-phosphatase, and buffer solution.  I began by isolating the dephosphorylated HipA using a Ni-affinity chromatography column.  After the protein was isolated, I needed to concentrate it up because a specific concentration is required for the phosphorylation assay.  I centrifuged 3 mLs of the elution fraction at a time, concentrating the protein.  Next, I needed to buffer exchange since the buffer used for the dephosphorylation reaction is different from the buffer required for the phosphorylation assay, so I centrifuged the protein three more times, diluting it by a factor of five with the new buffer each time, resulting in a 125-fold dilution.  I checked the concentration of the protein and found it to be 0.71 mg/mL, indicating approximately 70% yield, which is pretty good.  I left my protein in the fridge and headed to lunch.

After lunch, I began the phosphorylation assay.  I have already done this assay twice, but today I needed to try it with 10% DMSO to make sure that it gives the same results as without DMSO.  The compounds that I will test later are kept in 10% DMSO, so it is important to make sure that this will not affect my results and that the usual assays still work with DMSO.  I diluted my protein to a concentration of 0.05 mg/mL in assay buffer and put 500 uL of this dilution into one tube, and 450 uL of this dilution into a second tube.  I also added 50 uL of 100% DMSO to the second tube to create a concentration of 10% DMSO to mimic what the compounds to be tested later are stored in.  I removed 20 uL samples from each tube to serve as my controls.  Then I added 5 uL of ATP to each tube and incubated them at 37°C, beginning phosphorylation.  I removed 20 uL samples from each tube after five minutes, fifteen minutes, thirty minutes, forty-five minutes, an hour, and two hours.  After removing the samples, I immediately heated them at 99°C for five minutes to denature the protein and stop the reaction.  I then added loading dye and stored them in the fridge for later when I will run the gel that will show me my results.  The purpose of removing samples at different times is to demonstrate how HipA auto-phosphorylates in the presence of ATP over time.  I will compare the results of the protein with and without 10% DMSO to determine if the DMSO affected HipA’s auto-phosphorylation or if it affects the phosphostain procedure that I will use to visualize the gel.  If the two gels look the same, I am safe to use this procedure with the test compounds.  If the gels do not look the same and the DMSO does in fact affect the assay, I will have some troubleshooting to do before I can test out the compounds.

Overall, a usual, but rewarding, day in lab.  I can’t wait to stain the gels and see what my results are.

Sincerely,

Caroline

All Clear on the Western Blot

It’s always a great day when the long-awaited results of my western blot come back clear and easy to interpret. My project this summer involves many, many western blots. Western blot is an important technique used to detect proteins in a sample. To start my project, my mentor and I dissected samples of different regions of mice brains at different time points in development from both our mice model of dystonia and its littermate controls. I am now using western blot analysis on these samples to determine if there is a dysregulation in the levels of phosphorylated eIF2alpha (our protein of interest) when comparing the mice with dystonia to the normal mice. 

Though the process of western blot remains the same, my day to day in lab changes based on what step I am at in the process. The whole process of western blot from start to finish typically takes about 3 days. Though it takes a while to get results, it is very rewarding when it works correctly. The process begins by first homogenizing the brain tissue samples and adding buffers to ensure the proteins remain in tact. This step is done in the cold room to ensure the proteins don’t denature. I then do a BCA analysis to determine how strong the protein concentration is in my sample, so I know how much sample to use when I run the gel.

The next step is gel electrophoresis, which helps separate the proteins based on their size using an electric current. After the gel is run, the gel is then blotted onto a solid support membrane to further analyze the proteins. In order to prevent nonspecific binding of the antibodies to the membrane, I add a blocking buffer to the membrane to block out any nonspecific spots on the membrane. To visualize the protein of interest, I then probe the membrane with a primary protein-specific antibody. The primary antibody binds to the protein of interest like a lock and key. I then probe the membrane with a labeled secondary antibody used for detection. I then use imaging to detect the protein-antibody-antibody complex on the membrane. Finally, I analyze the results to ensure the presence of a protein of interest, the amount of protein, and its size. 

Now that I am finally starting to understand the process and complete it primarily on my own, I come into lab everyday excited to learn from a previous blot. I love having the ability to implement better technique each time I complete the process. In addition, I am constantly learning new and better ways to complete each step from my lab mentor and other members of my lab. My lab has been incredibly welcoming and always willing to answer my questions. Though the process of western blot can seem a bit tedious at times, I am grateful to have the opportunity to keep learning through the process and be surrounded by such a supportive lab environment.

New day, fresh page.

My favorite part of life in the lab comes at the start of every day, when I get to flip to a fresh page in my lab notebook. I’ve always loved recording things, so you can imagine my excitement when I got my very own super-official-looking lab notebook, with its pristine black hardcover and exactly 100 pages of green-lined graph paper, each blank page promising a new scientific adventure. 

My lab notebook has become home to protocols, experimental set-ups, data, and observations. More importantly, it has become a way for me to reflect on the successes and failures of my experiments. While each day is inevitably different from the last, my days in the lab–like the pages of my lab notebook–follow a similar pattern.

Objective. Each page of my lab notebook begins with a few notes on my goals for the day, which I discuss with my mentor, Hailey, first thing in the morning. It’s often easy for me to get lost just doing the motions–pipetting and running gels while losing sight of the big picture. Setting clear goals at the beginning of each day has become an important anchor for me this summer, a way to remind myself of how each experiment contributes to the goal of my overall project.

Protocol. In the “Protocol” sections of my lab notebook, I record exactly what I do throughout an experiment, whether that’s setting up a PCR or transforming E. coli. Sometimes, this part of my day consists of pipetting until my hand starts to cramp up. Other times, it consists of setting up a gel and holding my breath as the computer loads the image of my gel to reveal the success or failure of a day’s work.

Results. Here I include observations or pictures describing the results of my experiments. Sometimes with sad face doodles, other times with an excessive number of exclamation points. At the end of each day, Hailey and I discuss our results and what they mean; we troubleshoot and plan for the next day.

Despite the loose structure of every day, I love the dynamic nature of life in the lab. I love that I don’t always know what my week will look like, because each experiment depends on the results of the last. I love the anticipation of walking into each day not knowing whether my experiment will work or not. And from that anticipation, I learn to value my failed experiments as much as my successful ones–each have equally important places in my notebook.