Ben’s In2Science blog
By Benjamin Simpson
Monday 15th August
When I arrived at the lab, the first thing I noticed was how casual everything was. Even the principal investigator arrived at about 11. I expected to find a strict regime of when/what to arrive, do and wear. I met with Niki who I was shadowing and Anna who was another scientist in the lad that I spent a lot of time with. There was also another in2science student in the lab called Afnan who was Anna’s shadowing student. Most of the experiments and work, we did together. Anna was using Zebrafish to investigate the development of myelin (A substance which is wrapped around neurons to increase conductivity). Zebrafish are not regulated by ASPA under five days from fertilisation. They were especially useful because they are transparent allowing us to view the development under a microscope without harming the fish/embryo. On this first day they were only a few hours post fertilisation and so were still just a bundle of cells on a yolk. I learnt how to maintain the embryos through filtering out the dead ones and changing the water. After this we made internal and external solution for maintaining brain slices, with Niki. She is using rats and mice to research the effect of opening /closing ion channels in oligodendrocytes on levels of ca++. To do this we mainly calculated the quantity of the solution or compound necessary for the solution. We added glucose, various ions etc.
Figure 1: My lab drawings at 0 hpf
We spent the morning with Niki patching oligodendrocytes. We would incubate the brain slices for eight minutes. Then apply the electrode and monitor the Ca++ levels in the cell. The drug we were using (which affected the TRPA1 channels) would then be injected through the electrode and we could monitor the difference it made. Niki explained to me how to read the graphs and what to look out for in a “good” cell. Data of course needs to be very clear and conclusive in order to evidence a paper. We took several good results from the controls however the ones with the drug seemed to have higher starting Ca++ which meant that any rise that took place was smaller and less clear. We found out at a later date that thaxagagin, one of the drugs we were incubating in was a TRPA1 agonist which had caused the higher starting Ca++. Anna also took us to the area where the fish were kept. The scale on which they were kept was astounding. There were over 1500 fish in the facility. It was not dissimilar to a library but with tanks of fish instead of books. They were labelled with Wildtype of whichever adaptation they had. We were taught to tell the difference between the male and female fish. The females are a slightly different colour and slightly fatter with a tube to deliver eggs. We put the fish into pairs of a male and a female of the correct genetics so that they would lay eggs. Anna had collected some eggs that morning so we viewed them to see them at the six hours post fertilisation stage. By this point they were at various stages of covering the yolk, which is represented as x% epiholy.
Figure 2: Lab Drawings at 6 hpf
In the morning I continued the experiments with Niki. Though we used a different drug as to give better results. One of the tasks I was assigned was to make the electrodes. This was done by inserting a capillary tube into a machine which would heat and pull it into to electrodes. I had been told to do this manually, using a Bunsen, before in class (to little success). Now seeing how it was done properly made more sense and was highly effective. Today the fish had changed considerably and were developing organs. I was shown how to dechorionate them. Chorion is the bubble they grow in up to this point. Dechlorinating them is there for simply hatching them. This is done under the microscope with two sets of tweezers. I used them to pull the chorion apart and allow the fish out. This is beneficial for the fish and us as we can see them more clearly and the fish is free to move. Some of the fish had genes to be fluorescent in certain wavelengths of light, so we observed them in this light but they had not really developed enough to see much. There was a green haze around the heart and a red haze around the otoliths. Otoliths are the two dots in the ball behind the eyes. They are used for navigation and telling which way round the fish is. The blood flow was also visible though primarily used for transporting nutrients. Eighty percent of the oxygen required by the fish can be gained through the skin as at this stage as the surface area to volume ratio of the fish is very high.
Figure 4: Machine for creating electrodes
Figure 3: Lab drawing at 30 Hpf
Thursday was A level results day so I couldn’t be at the lab until around lunch time. (I only had one result to collect which was an A which I was happy with). The fish had grown but not vastly changed. In order to observe them we had to anaesthetise them and mount them. To do this we placed them in an agarose gel solution which would set and hold them still. Despite its simplicity it was a surprisingly difficult task. To line up and orientate the fish (in the small amount of time before it set) we had to use a pipette tip and microscope. Bearing in mind that even then the fish were only 2-4 mm (my estimate). I managed to do six on a slide. Anna however was doing twelve at a time with even greater accuracy. The fluorescent fish had also grown and become a lot clearer. The heart was now incredibly clear. I could easily see the chambers and valves. The red was showing myelinated areas which were now the otoliths, jaw, fins, and lateral line (grows above spine).
The Fish were now large enough for the planned investigation. Therefore, we mounted them as yesterday. I taught Afnan how to do so. Then we screened them. This consisted of viewing each fish beneath the microscope (confocal) and seeing which ones had the best developed nervous systems and would work best. It would be noted which ones were worth using and they would be remounted later for use. We screened seventy-three fish and found five that were good enough to use. After lunch Mark arrived at the lab who Anna was collaborating with for the investigation. Two electrodes were inserted onto the spine of the fish, one near the head and one down on the tail, then a needle into the brain of the fish. The first electrode was to send a signal down the spine and the second to record it. (Stimulating and recording electrodes). We took the heart rate of the fish before and after injecting the fish with TTX, The drug in puffer fish. This was to check that the fish was still alive and wasn’t experiencing any other effects. We were looking for the impulse sent by the stimulating electrode to no longer be received by the recording electrode one the TTX was introduced. This is due to the TTX closing the Potassium channels in the neurons and preventing the impulse from being transmitted. It worked fantastically. They were absolutely ecstatic and celebrating at first whilst I was staring at the graph working out what had happened but I quickly caught on. By this point it was nearly nine o’clock. One of the disadvantages of the relaxed work style is that people turn up in the afternoon and work can easily go on until midnight. Some of the days even for me were quite long. On the upside, you can take lunch whenever suits you and there’s far more freedom. The work seems to be heavily driven by the need to finish a paper or acquire a grant. The self-motivation suited me well. The phrase “Publish or perish” was extremely relevant. Job security seemed to be an issue.
Figure 5: Me observing the fish
Niki informed us that we would be doing a clickit experiment in the next week. Therefore, I spent the morning calculating the various quantities of solutions and substances that needed to be put into solutions and added to our internal and external stock. I also familiarised myself with the procedure. (Which I will go through the stages of on the days that I do them). In the afternoon I Helped Niki with data analysis of what we did last week. Which consisted of taking the section of the graph from before and after the addition of the drug. Using a template to extract all of the impulses. Removing the outliers and all the electrical noise. Then taking an average to see a simple before and after. The data showed what we wanted but it was not very clear cut. Niki thought that she would have to take more results to evidence her point.
We made up the necessary solutions and labelled them as follows: WT PITC, WT Control, KO PITC and KO Control. PITC was the Drug that we were using and the point of the experiment was to find out where it stuck to on the brain slices. There was also a dye attached to it with a copper catalyst, glucose, ions etc. in the solutions. WT represented the wild type mice and KO was the knock out. Which was the mice with the TRPA1 channels removed. TRPA1 was where we expected the PITC to stick to. (Niki had previously done the investigation.) She had not done a control though so those results were the ones she was most interested in. The mice we used were one hundred and ninety days old which is quite old for a mouse. After we had made the brain slices we left them in the four solutions for an hour with isolectin, a dye. We had six slices of each variant. Then we placed the slices in a 4% paraformaldehyde solution for another hour. This is to fix the slices. The PFM kept everything in the cell the same over the next few days as we view them. Then we washed the slices three times in phosphate buffer saline (PBS). Next we added a solution containing Tritin and Normal goat serum. The tritin was to permeablise the cell (make holes in it) and the NGS to remove the various antibodies that would hinder the investigation. The clickit solution came next. Containing the reaction buffer, Copper sulphate, additive, and chomeo 543 (A dye). We washed it again and added DAPI (a nuclear dye) but by this point it was getting late so we covered it over night to mount the next day.
Figure 6: Me taking photos using the microscope
We began around ten o’clock as normal by visiting the fish again to clean and observe the next batch. They were the same as the first and we were getting far better at it. This time we took the photos at each stage using a Hubble microscope. The ones I have included so are some of these. After lunch we mounted the slices we had prepared yesterday. (After a wash in PBS) To do so we used a spatula to move the slice into a drop of PBS on a slide. We used a pair of tweezers to unfold and orientate the slices on the slide before drying them by soaking up the PBS with a piece of tissue. Before placing on a cover we added a drop of mounting solution. The covers were lowered at an angle to avoid air bubbles. We then left the slices over night to image later.
Before viewing the slices under the microscope we sealed around the edges with clear nail varnish, simply to make it air tight. Ironically I was exceptionally good at this. I have had plenty practise painting though, just not with a nail brush. I think that having this steady hand helped in a lot of the things I did in the lab. We viewed the slides at X20 and the three dyes could clearly be seen. Chromeo 543- Red, Dapi-Blue, Isolectin-Green. Unfortunately the where the PITC was sticking wasn’t entirely clear. However we found the next day that at X63 it is far clearer and what we expected. The images were however absolutely stunning, even at the lower magnification. I also briefly observed Anna and Marks experiment with the TTX and the Zebrafish again. In the evening there was a celebratory gathering out on the main quad which I was invited to due to the publication of some of the researchers in the lab’s paper in the Nature journal (which I gather is a journal of great renown thus making it a big deal). The authors were there and It was nice to get to know everyone at the lab better.
I spent most of the Friday working with the data and images I had collected. I also took some more photos of the Brain slices at a magnification of X63. We also took the final photos of the fish and had a competition between me and Afnan as to who could correctly mount more fish on three slides as described earlier. I won by one fish which was extremely close and greatly amusing. I stayed late and gathered more photographs for the competitions and to say my goodbyes. I am very much grateful for the experience and took a lot from it. There are so many small things that you learn in class that seem to make little difference but out in the industry are absolutely vital. My thanks to Niki, Anna and Dave Atwell (the principal investigator who I haven’t really mentioned but was very nice and I got on well with).