in2scienceUK blog by Adeolu Busari
Despite the warning at the introduction day that the labs we will be entering will not resemble ‘a giant room filled with new state of the art tech and machinery littered with scientists in their lab coats constantly pipetting’. I still had small hopes that some aspects of the description would be true.
How I was wrong.
On the first day I was nearly convinced that Dr Hristova’s lab was a kitchen. There were 3 fridges, 2 weighing scales, an oven and a microwave (definitely a kitchen I thought), but then I snapped back into reality once the fridge was opened and I saw it packed full with all these different chemicals and the huge bottles of methanol and ethanol in the cupboard with the bright orange flammable symbol on it.
Despite the fact that the atmosphere and the appearance of the lab was different from what I anticipated, I’m glad it was. The lab had a friendly, warm atmosphere and was hardly ever packed. The masters’ students and Dr Hristova were friendly and would often engage me in conversation and tell me about their project.
Now onto the actual science: The project outline at the lab I was placed at was to study the effects of pharmacological inhibition of TGF-β1 on microglial cells after mild neonatal hypoxic ischemic insult. The study comes from unpublished data which has found the deletion of TGF-β1 in microglia to be protective. The aim is to see if pharmacological inhibition of TGFR-1 would prove neuroprotective.
On the first day I observed images of stained mice brains underneath a microscope, these images showed areas of the brain that had some physical damage to it. This damage was due to hypoxic-ischemic event, meaning the brain was partially oxygen deprived and that brain cells died. We measured the amount of physical damage noticed in them, physical damage meaning that the brain matter had been fragmented due to the dead neuronal cells.
The remaining of my two weeks in the lab I helped in prep for staining and staining the brains, preparation consisted of taking out frozen sections of neonatal brains and spreading them. Spreading is a process where the brains are rehydrated with bidistilled water (ddH2O), then spread using fine brushes to become flattened and fan dried at room temperature.
Later the sections were fixed in 4% formaldehyde and washed in 0.1M phosphate buffer for 5 minutes. Then defatted in acetone for 2 minutes each, washed twice in 0.1M PB, and then in 0.1% bovine serum albumin/0.1M PB (PB/BSA). After, they were placed on wet chambers and blocked in 5% goat serum in 0.1M PB for 30 minutes. The block was removed, and 90µL of primary antibody was applied to be kept on overnight.
On the second day of fixing, the excess primary antibody was washed off using PB/BSA, PB, PB and PB/BSA and a few more washes took place to insure there were no more reactions taking place.
Finally the sections were ready to be stained either via nissl or tunel. Tunel is a stain that binds to fragmented DNA, so is a specific marker for cell death. Whereas nissl stains cell bodies. If a cell was damaged it would not stain much with the nissl, it may not even stain at all. This is why two stains are used whilst analysing the effects of hypoxic-ischemic even on neonatal brains.
In order to stain via tunel the sections were spread and fixed as described above and then incubated in 3% H2O2 in methanol and then washed in 0.1M PB. Sections were incubated with 0.1% terminal deoxytransferase and 0.15% deoxyuridine triphosphate for 2 hours at 37°C. The reaction was stopped in tunel stop solution for 10 minutes. Then lastly the sections were washed thrice times in 0.1M PB and incubated for 1 hour in ABC solution.Nissl staining is not that different to tunel but is also complicated, so I will not be describing it due to the word count limit!
Overall, I really enjoyed my lab placement and I feel very fortunate to have been allocated it. It has allowed me to witness the research side of science, which is far more interesting and practical than I thought it would be. Thank you Dr Hristova and In2science for giving me the opportunity.