Part II: Regina’s second week at KCL

My second week at King’s was spent with Dr Matthew Grubb and his team consisting of Elisa Galliano and Adna Dumitriscu which I got to work with closely with. Their project was more focused on neurons and more specifically, the olfactory bulb in mice. They want to see if and how stimuli affects the AIS (Axon Initial Segment) in neurons where action potentials are generated, whether it affects its length and how far along the axon it is. This can be done with a process called patching where you can observe the action potentials and the way the neurons interact with each other.

I wasn’t able to participate as much in this week as most of the things were technical but I was able to watch and help out in small ways, all the while asking questions about their work. I was able to use a confocal microscope to view some of the zebrafish slides after some immunofluorescence.  This works by targeting antigens with a primary antibody and the fluorophore as a secondary antibody to target that primary antibody so we are able to detect the antigens which in this case is the TH enzyme (tyrosine hydroxylase) in the olfactory bulb. The AnkyrinG also found in the AIS is what enables us to see the latter as they’re the ones being labelled.

 Here is a picture of me using a confocal microscope and seeing some dopaminergic cells found in the CNS and shown due to some immunohistochemistry!

Here is a picture of me using a confocal microscope and seeing some dopaminergic cells found in the CNS and shown due to some immunohistochemistry!

It was a real eye-opener to work in this lab, the two weeks felt really short but I was introduced to so many aspects of neuroscience and how life as a scientist would be so I’m really grateful for this opportunity and that I was able to meet such amazing people. Everyone was so passionate about their project and so eager to help me out in introducing their work and making sure I was able to make the most out of it; I could write about 20 blog entries!

Thank you to In2ScienceUK and everyone at King’s!

Part I: Regina’s placement in Neuroscience at KCL

by Regina Gul

My placement this summer was at King’s College London in the Neuroscience department for the duration of two weeks (17th August – 28th August 2015). Before I start this entry, I would like to mention just how wonderful everyone was at making sure I was well accommodated with their work, that I knew what was going on and that I was as involved as possible to make the most out of this opportunity. With that said, I’ll start off talking about my first week which was spent with Dr Caroline Formstone before writing another entry about the work I did the Dr Matthew Grubb and his team (shout out to Elisa and Adna!) during the second week.

Dr Formstone is interested in how the early CNS was formed, the formation of neural tubes, how neurons set up their circuits and If neuroepithelial cells have polarity systems that allow the neurons to move towards the same place constantly.

Neurons move down the cerebellum in the back of the brain and what Dr Formstone is trying to find out is if this movement is generated by the neurons themselves or by the neuroepithelial cells. These cells have adhesions between them, both at their apical and basal ends to make sure that nothing gets through and it even allows the epithelial cells to communicate with each other. Now, the protein found in the adhesions varies – there is a variant 1 and variant 2. The differences between these two variants are that after the transcription of RNA, the last exon shown is either E14 or M14. The ratio of E14 to M14 is general 50:50 but this can be manipulated to get more or less of one. With this, we could predict whether it is the protein that causes the neurons to migrate towards the tail, whether it is this “parental guidance” given by the epithelial cells that leads them on.

RG1

With these different honing systems, it means that these variants are placed at different parts of the cell. But this is just a hypothesis, even though we know there are different proteins at the basal and apical ends of the neuroepithelial cells, we cannot be certain of this.

So in my first week, we wanted to carry out the project that the students from In2Science had previously started with her. What they had done is that they had managed to get onto the stage where they amplified the last exon (E14 or M14) using a PCR (polymerase chain reaction) and cloned it into the plasmid vector which is specifically constructed to allow protein expression at high level. The variant 2 of PDZ domain motif is found in proteins as a way of targeting a protein to a particular part of the cell and what we’re trying to find out is if the PDZ domain targets a particular part of the cell and what proteins are involved in the distribution process. Essentially: does it organise the migration of neurons down the cerebellum?

Throughout the week we grew agar jelly and BL21 bacteria to their exponential phase (when they grow fastest) and DNA as well as a digest so we could run this on a gel. The gel is mainly to help us ensure that the DNA is fine; it creates a ladder due to a marker and then when the DNA is added you can see the different bands and how much of it there is. We also added IPTG which helps transcribe the RNA which can then be translated as a protein and so we added this into some cell membranes with GFP (green fluorescent protein) to allow us to later see the green when left overnight. About 3/20 cells showed the GFP tag!

RG2

My first week was really interesting and fascinating, I learnt to see just how life as a scientist would be and got to be in an environment where I used many machines and learnt about simple lab skills that you can’t just learn out of a textbook. Things like using a shaking incubator, a centrifuge, running a gel, growing agar jelly, growing bacteria, or even simply pipetting with different equipment than we do at school became ordinary!

Part II: Tissue Culture

by Hafiza Irshad

During my two weeks in the lab I was given some cells to look after (A549), I developed a strong bond with these cells over the two weeks and loved them as my own. The cells divide constantly, if they outgrow their container they run the risk of dying, something I definitely didn’t want happening. To avoid this I ‘split’ the cells every two days. I was able to tell if the cell required splitting by tracking how confluent (closely packed) they were by looking at the cells through a microscope.

Blue coats are worn in tissue culture labs only, this prevents the cells from being exposed to dangerous chemicals we use in the lab. The hood provides a sterile environment.
Blue coats are worn in tissue culture labs only, this prevents the cells from being exposed to dangerous chemicals we use in the lab. The hood provides a sterile environment.
A pellet containing cells is visible at the bottom of the tube, and the pink medium above will be discarded
A pellet containing cells is visible at the bottom of the tube, and the pink medium above will be discarded

The splitting was done an aseptic hood in sterile conditions. I would extract the old media, perform a PBS wash and add an enzyme known as Trypsin (to ‘unstick’ the cells from the bottom of the flask). The cells would then sit in an incubator at 37° until they were visibly moving. I then add extra media and ‘spin’ them down in a mini centrifuge. This would create a pellet of my cells so I could tip out the media and then suspend in fresh media. I would then transfer a third of this liquid into a fresh flask and get rid of the rest. This means there is a third of the amount of cells previously in the same sized flask. The cells have more room and are able to live for another day or so before they require splitting again.

Tissue culture was challenging because of the strict sterile conditions involved, if your pipette touched anything other than the liquid you intended on measuring, you would have to dispose of it and get a new one. There are also different methods that people follow for splitting cells, for example, a PhD student in the lab told me I wouldn’t need to use the centrifuge if I added extra Trypsin. This method worked for him but my supervisor Laura had always used the centrifuge. It all depended on who taught you first and what you feel comfortable doing. I really enjoyed working in the tissue culture lab and looking after my cells (which I have been told are now frozen down for use later).

Lab work with Laura at Imperial College London

by Hafiza Irshad

The two weeks I spent in lab were the highlight of my summer. The equipment surpassed anything we had in school labs. No squeezy pipettes, no ball pipettes – which never seem to work and amazingly, I didn’t have to wear goggles (I was safe without them).

Aside from all the luxuries I experienced from being in a legitimate laboratory, the learning experience was incredibly satisfying. Doing my EPQ project on cancer, I thought I had a fairly sound knowledge on the development of cancer, however within the first hour of my placement Dr Laura Yates had proved me very wrong indeed.

One of the first signposts of cancer is that the adhesion between cells is lost. This enables cells to migrate to other parts of the body and possibly form tumours. The work I did with Dr Laura included staining slices of lung tissue to identify the strength of the proteins that are responsible for cell adhesion.

The first protein I stained for was E-cadherin. The protocol was fairly simple but each step was very crucial to be able to get a good stain. For example, one needs to incubate the tissue in a ‘block’ to prevent non-specific binding to any other proteins. PBS (a water based salt solution used as a buffer) washes are done after each block followed by incubation to get rid of any background staining that may prohibit the visibility of the protein stain. I repeated the process on different samples of proteins – Vangl2 and MMP12.  The protocols were almost identical for all but had slight differences. For example, Vangl2 required a stronger, additional block and the secondary solution used differed too.

This is the tray I used for all the staining I did.
This is the tray I used for all the staining I did.

To get lungs thin enough to stain and observe they must be sliced. This sounds perfectly simple but it was something that definitely required a lot of experience, a gel is used to stick the sample to a block of wax, then cooling the gel until it’s hard using an ultra-cold covering. Next you place this section of lung into the slicing machine and with each slice made, you retrieve from the media (liquid containing nutrients for the cells) placed in a small tub underneath. You then place these super small slices into wells filled with fresh media.

Sample of lung tissue to be sliced. It is far too big for staining.
Sample of lung tissue to be sliced. It is far too big for staining.
Slicing machine
Slicing machine

This is what one of the slides I stained looked like under the microscope:

fig4

This branch of science is called “immunohistochemistry” and is all related to the detection of proteins within samples. Staining is an important technique used in research as it helps you prove (or possibly disprove) your theories and allows you to share your work to other scientists. I enjoyed staining because there are many techniques involved, teaching me lots in a short amount of time.

Oil Red O Staining at the Lungs for Living Research Centre, UCL

by Charlotte Loos-Bennett

fig1While working in the Lungs for Living Research Centre at UCL I assisted with various experiments involving cellular biomedicine; particularly assessing ways to prevent the diminished function of the thymus (a lymphoid organ which produces T-lymphocytes for the immune system) with age. One said experiment was Oil red O staining.  The aim of this was to identify adipose tissue and distinguish between transgenic tissue (genetically modified or mutant type) and the wild type tissue (from individuals innatural conditions). The tissue for the slides was taken from a mammalian thymus.

For this experiment we were given 6 slides, four of which are from old thymus, and two test slides. Those from the old thymus (O series) acted as control slides and were expected to stain correctly. This is to represent how in the ageing process the thymus is infiltrated by adipose (fatty) tissue and undergoes thymus involution, where the thymus undergoes a progressive reduction in size leading to a reduced immune system. The other two slides (B series) were the test slides and were either a transgenic or a wild type. The slides had previously been fixed. The purpose of this was to make the tissue rigid.

fig2fig3

Firstly, we rinsed the slides with 60% Isopropanol (300ml isopropanol, 200ml water) for 2 minutes before draining the slides. We then rinsed them with the Oil red O working solution (50% Oil red O with 50% water) for 15 minutes which would stain the adipose tissue red. Following this, a further rinse with Isopropanol was undertaken. To aid the rinsing of the slides they were placed on an orbital shaker. The slides were then dipped 5 times consecutively in the stain Haematoxylin (which stains nuclei blue) and rinsed in distilled water for 5 minutes. The final step in the staining process was to administer some Mowiol mount and cover the slide with a cover slip, ensuring there were no bubbles.

fig4When viewing, under the microscope and then using a Nanozoomer, each of the O series you could see how the adipose had infiltrated the thymus. For example O1 there was nothing but adipose tissue but O2, while there was still adipose; inside on half of the sample we observed blue (the stained nuclei). We concluded for O1 that you could only see red as when the slide had been cut; it had not gone deep enough into the thymus to get any of the actual tissue, as opposed to the adipose tissue on the surface. Once we had established the control we needed to identify which (from the B series) was the transgenic and which was the wild type. Upon observation we concluded it to be B2 that is the wild type. This is because B2 contained more blue and was therefore a much healthier tissue. B8 was a mutant as it was not packed with enough blue due to the lack of sufficiently healthy cells. While both samples contained adipose throughout it was the lack of healthy cells that hinted it was mutant and therefore fulfilled the aim of the experiment.

How is your personality linked to the way you write stories?

By Amy Matthews

My placement was at the Institute of Cognitive Neuroscience at UCL. We have been investigating whether there is a link between personality traits and the way people add emotion to neutral stimulus.

We investigated this by recruiting participants who would be the writers of stories about neutral words; before they began to write stories they were given the Big 5 Inventory personality test. This tested participants on the five main personality traits: openness, conscientiousness, extraversion, agreeableness and neuroticism. The participants were told to work with the first idea for a story that came to them and that the stories had to be at least three lines long.

A second group of participants were also recruited who would rate these stories for creativity, arousal and emotional valence. This group of participants were given 25 randomised stories written by the other group of participants; however they did not know that they were written by previous participants.

fig1

In our results we found a surprising link between openness and creativity, open people are usually more curious, original and open to new ideas. However our results found that more open people wrote less creative stories. An explanation for this is that more open people are open in real life situations rather than in fiction, whereas less open people are not creative in real-life so they compensate for it in fiction.

fig2

Also from our results we found that as expected, the participants rated highly for neuroticism wrote more unhappy stories. People who are rated highly for neuroticism are more anxious, irritable and temperamental. This means that the stories that they wrote were more unpleasant than other participants, showing that there is a link between neuroticism and creating negative stories from neutral words.

fig3

Overall, there is a link between personality traits and people’s perception of neutral words as two traits showed a strong link, however not all personality traits were reflected in how people wrote stories about neutral words.