Science Diaries: The final lab week

Science Diaries: The final lab week

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Since my first blog post “Are you what your mother ate?” I’ve been working like crazy in the lab to finish my final experiments for my entire PhD (absolute madness!), and after over three years of PhD life and hard work the final lab week is here!

One of my reasons in making this blog is to give others an insight into the life of a PhD student. When I applied for my PhD I was currently doing my masters project but knew a PhD was going to be a big step up. I wanted to understand more about what it was like to do one, but couldn’t find many people’s experiences documented online. So here is is, one day in the life of one PhD student…

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Wakey wakey

After pressing snooze on my alarm a good few times and being too cosy to jump out of bed, a typical day for me starts at 6:45am. I get up, and as it’s December I go and find my advent chocolate which makes getting up that little bit easier! Porridge is always my staple breakfast as it gives me a good amount of energy to start a day in the lab. My walk into work is about 20 minutes which is great – not too far away but enough time to get fresh air and clear my mind before I’m deep in antibody calculations, immunofluorescence staining, PCR analysis and microscopy. A LOT to do!

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Work begins

Works starts nice and early at 8am (got to be productive before the necessary tea break)! First things first, check my to-do list that I made at the start of the week. Top tip – being organised and planning your week is a massive help to keeping on track and being productive amongst the craziness of a PhD. The last couple of months I’ve been doing a lot of immunofluorescence staining so I check my plan and grab the right set of samples out of the freezer and let them air dry before I start the experiment.

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Getting prepped

Whilst waiting for the samples to dry I check emails, calculate antibody dilutions and prepare the necessary buffers to use for my immunofluorescence staining.

What is immunofluorescence staining?

So my project is all about looking at how the offspring’s skeletal muscle development and function is affected by a maternal high-fat (first study) or vitamin D deficient (second study) diet. Research shows that diet can alter the type of muscle fibres that make up an individual’s muscle, so this is what this experiment is for. As a brief insight, I have cross sections of muscle samples previously cut and put onto glass slides (as you can see in the previous picture). Different muscle fibres express different types of the protein myosin heavy chain (MHC). Adding antibodies specific for these proteins and then adding a fluorescent tag to each one allows me to see which fibres are what type under the microscope, and ultimately see how the number of each type of muscle fibre changes with a normal or altered diet.

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Primary antibodies on and two hours to wait!

These primary antibodies are what attach to the specific types of MHC protein. I make two different cocktails of antibodies which are mixed with either of the two different buffers made above. One muscle sample will be incubated in three out of five primary antibodies, and another section of the same muscle will be incubated with the other two antibodies. I also have to include ‘negative controls’, these are sections of the same sample which are incubated in just the buffer without any antibodies. As there are no antibodies applied to these samples, in theory they should not fluoresce when I image the samples, and it shows that any fluorescence is due to the primary antibody attaching to the correct molecule.

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Tea break o’clock

The 11am tea break is a fundamental part of my day! My friends in the lab and I pretty much always have this without fail. Bit of caffeine and a catch up means a boost of energy and ready to go… until lunch break!

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Catch up take one: PCR analysis

Whilst waiting for my two-hour incubation to finish I catch up on other bits of work. Here I’m analysing a PCR I did a couple days before. PCR (polymerase chain reaction) is a technique that allows me to quantify how much of a specific gene is expressed in my tissue samples. Here I was looking at the gene troponin 1 – a gene important for the expression of a protein involved in muscle contraction regulation. I’m seeing a few significant differences (yay!), a point where all PhD students have a feeling of relief!

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Primary antibodies off, secondary antibodies on

After two hours, the primary antibodies are washed off with the buffers and the secondary antibodies are applied for one hour. The different secondary antibodies recognise their corresponding primary antibody and bind to them. They have a fluorophore attached and this is what makes the muscle fibres a fluorescent colour when I look at the samples down the microscope. It’s important that these are incubated with a non-see through lid as exposure of the fluorophore to light will ruin the fluorescent signal. Too much exposure to light = experiment fail.

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Catch up take two. Making pretty pictures

After my secondary antibodies have been applied, washed off after an hour and stored away ready for image analysis the next day, I catch up on some remaining image analysis from the previous day. This is all done in a dark room – how wonderful! This picture shows a typical muscle cell stained for the fibre type IIa. Using the fluorescent microscope, I change the filters to view the other fluorophores applied to the sample and therefore see the other fibre types which fluoresce in different colours. I use green, red and blue, which means pretty looking pictures!

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Me time = crossfit time

Lab work for the day done and off to crossfit I go.

A work/life balance is extremely important to me, and exercise is essential. If possible I go every weekday evening. Working out helps me stay healthy and gives me that time away from the lab to focus on something else, and relax.

After crossfit, it’s time to meet up with friends or go home and chill with some food. Day done!

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There it is, a snapshot of one day in the lab. Admittedly I am in my last week but this has been a pretty typical day for me over the last couple months in between other experiments going on. Of course a day in the lab can be completely different to this. I had days where I’d be doing long 18-hour days due to animal studies, or shorter days in my first year when there wasn’t as many bits and pieces to do. But for those who don’t know what kind of thing us PhD students get up to, I hope this gives you a bit more of an idea!

So my lab time is up, and off I go to enjoy my Christmas holidays before I start the mammoth task of writing my thesis.

Merry Christmas everyone!

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