Learning Immunohistochemistry: More Than Just Pretty Pictures

I had expected the initial few weeks of my time in the lab to be fairly challenging to say the least. As an undergraduate, my typical experience of being in a lab environment is a three hour frenzy of activity, frantically trying to get everything done in the allotted time before I begin to feel bad that I have managed to keep the demonstrators in the lab past five o’clock. Moving to a working lab therefore, where people do research day-in day-out, I was a tad worried how different an environment it would be, where what we are trying to found out hasn’t been verified dozens of times by students doing the exact same practical the year before. However over the past month, my confidence has grown tremendously as I have had the opportunity to practise and optimise my protocol for a common biological technique: immunohistochemistry.

This technique is used to visualise expression of a protein you are interested in, allowing researchers to observe expression in a target tissue. First an antibody, referred to as the primary antibody, is raised in an animal by injection of the target protein. It is extracted, and can then be used with the tissue we are investigating, as it will bind to our target. We then use another antibody called a secondary antibody, which binds to the primary antibody and has attached a molecule that emits light after excitation with a certain wavelength, which we can then visualise under a microscope.

This has been my main protocol so far during my time in the lab. I am investigating protein expression at points of communication between nerve cells in sections of mouse spinal cord. I am able to see the junctions between the cells, and other cells potentially involved in their communication and function under an Apotome microscope. Learning to use this rather expensive and intimidating-looking piece of equipment was initially scary, however being able to use it independently has been incredibly rewarding. When the immunostaining works as it should, it allows me to produce images that let me visualise individual synapses between cells, which is well worth the length and at times, unreliability of the procedure.

An example of an immunohistochemistry image. VGLUT2 (red) and PSD95 (green) are presynaptic and postsynaptic markers respectively. The white boxes highlight puncta which lie juxtaposed to one another, and therefore likely represent individual synapses.

Immunostaining takes 3 days from start to finish, and involves a large number of steps, so I am glad that this has been the main procedure I’ve been doing in the lab in that it has taught me patience. “That’s science” is a phrase that gets used a lot, and what it has taught me is probably the most important thing I will take away from this experience. The mild soul-crushing nature of seeing a week’s work gone in an instant when I go to image my tissue under the microscope and see, as is common with this method for a multitude of reasons, that no staining has occurred, highlights that working in science requires patience. Other members of the lab have said if anything, when things work first try, you become paranoid that somehow you have done something wrong. The reality of how long it can take to get any meaningful results is something you don’t experience in undergraduate practicals, and having the patience required seems to be very important. However I have also learned that while it can be frustrating, the satisfaction of something finally working is more than worth it.


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