Experimenting with Immunohistochemistry...

As I thought, this week has had a lot more going on! The lab staff has begun to give me more responsibility, and I have now begun my first protocol: a two-day Immunohistochemistry (IHC) experiment.

Autocorrect tells me "immunohistochemistry" is not a word. Trust me, it is. I would be just as lost if not for my internship in Dr. Gallitano's biomedical lab at the University of Arizona this past Summer. IHC, colloquially known as "immuno," is a process I am already very familiar with, and have performed before at the last lab under different conditions. Instead of using mouse brain tissue floating in solution, we are using human brain tissue that has already been mounted onto slides.

So what is immunohistochemistry? It is a process that allows us to stain certain proteins in cells such that they can be seen and documented. The best way to explain it is to break down the word itself. First, we have "immuno." Just as it sounds, this means IHC involves immunology, in this case, the use of antibodies (found in the immune system), that bind to certain antigens (proteins on the surface of or in cells). Next, we have "histo," as in "histology"- the study of animal tissues. So we are using immunology techniques such as the use of antibodies in animal tissues. Lastly, we have chemistry-- the chemical analysis of whatever tissue we are studying using this technique. In one definition, immunohistochemistry is the process of introducing specific antibodies to animal tissue in order to bind specific antigens, and thus be able to visualize those proteins under a microscope. Hopefully I haven't lost you yet.

The diagram above is very helpful. At my old lab, we did the protocol on right, immunofluorescence. At the Mufson lab, we do the one on the left, indirect immunohistochemistry. Both follow the same general principles.

You start with a protein you want to "label", the one you want to be able to visualize in the cells to confirm their presence (or absence). At this lab it might be plaques, abnormal clusters of beta-amyloid proteins that build up between brain cells and are characteristic of Alzheimer's disease. So, if you want to label for beta-amyloid, you have to introduce the antibody that will bind to it, anti-beta-amyloid. Because this is in humans, the antibody needs to be made in a different species such as mouse, so it will not bind to any other human proteins. This first antibody we use, the primary antibody, in this case would be mouse anti-beta-amyloid. In the diagram above, the primary antibody is in red. In the immunofluorescence protocol on the right, this antibody has a fluorescent tag bound to it, a protein that fluoresces under certain light. At the old lab we used green fluorescent protein (GFP) produced in jellyfish that indicated the cells which housed our target protein.

In this lab we need a secondary antibody that binds to the first in order to label our target protein. Because the secondary antibody is binding to the primary, it needs to be anti-whatever the primary antibody was made in. If our primary was mouse anti-beta amyloid, the secondary would have to be something anti-mouse. It could be goat, chicken, or horse anti-mouse, as long as it is produced in a different species and targets the mouse primary. In the diagram above, the secondary is in blue. Attached to the secondary antibody (represented by the pink circle) is a protein enzyme called horseradish peroxidase (HRPO). Yes, it is derived from the horseradish plant, specifically the roots. By itself, it does not do much. However, when we add diaminobenzidine (DAB) and some hydrogen peroxide (H2O2), the HRPO catalyzes a reaction between the two that produces an insoluble brown precipitate (colored solid) attached wherever your antibody has bound to your target antigen. You have labelled your protein!

The images below from leicabiosystems.com show actual indirect immunohistochemistry staining.

This image depicts beta-amyloid immunohistochemical staining of an Alzheimer's Disease affected brain. The brown staining of the plaques is very clear.

Similarly, this image depicts immunohistochemical staining in AD affected brain tissue for something called amyloid precursor protein (APP). Beta amyloid is produced as a fragment of this protein, and so the presence of APP is also considered a characteristic of Alzheimer's Disease, and is identified through IHC protocol.

I am conducting an IHC experiment that I will write about soon! I'll keep you posted.

Citations:

Brains and Brawn - Antibodies in Research and Diagnosis. (n.d.). Retrieved February 15, 2017, from http://www.leicabiosystems.com/pathologyleaders/brains-and-brawn-antibodies-in-research-and-diagnosis/