By Kathryn Ippolito, MPFI Postbac 2019-20
If you have ever spent time clicking through Google maps, zooming from a mile high to a street view, you have a good idea of how neuroscientists feel when they look at the brain. It is possible to see a few features of the brain with the human eye, such as major sections, known as lobes, or the wrinkles on its surface, known as sulci. However, in order to learn what happens at the cellular level—where each cell is located, how they interact, what happens inside each one when they are active—researchers have developed several methods to “zoom” in on particular areas.
Since the late 19th century, staining has been used to help make nearly transparent brain cells visible under a microscope. Scientists can apply the stain to a very thin section of the brain in a variety of ways. This process is called histochemistry. Different chemicals can highlight different structures within a tissue sample, from proteins to larger areas like the cell wall. Stains are commonly used to analyze cell structure and connectivity or to diagnose cellular abnormalities like those seen in cancer. In the past 30 years, immunohistochemistry (IHC), or the use of antibodies to target and identify specific molecules, has revolutionized the field of tissue staining. Antibodies are proteins naturally synthesized by the immune system as part of the body’s defense to foreign particles. Antibodies can bind to almost any compound, known as antigens. This includes proteins that only exist in specific cell types, which allows researchers to distinguish cells with internal differences even if they appear identical at first glance.
With a few steps, scientists can customize antibodies to go anywhere in the brain and express one of several colors when it finds its target. The first part of this process is selecting a primary antibody, which seeks the protein of interest. Next, it is essential to use a secondary antibody that recognizes the primary antibody and is also linked to an enzyme or fluorescent tag. That tag will visibly mark the area associated with the primary antibody when activated. This may seem simple enough, but there are several factors that scientists must keep in mind to ensure that are able to visualize the cells of interest with clarity and specificity.
Typically, antibodies do not naturally exist for every molecule being studied but can be produced by immunizing research animals against a specific antigen. Monoclonal antibodies are many copies of the same antibody pulled from one immune cell that has been cloned into a cell line. These antibodies only bind to one site on the antigen and very consistently label the correct molecule, but require a high investment of time and money. The alternative is polyclonal antibodies, which are essentially all the antibodies produced against a given antigen. They can be produced relatively quickly and interact with many binding sites, which increases the signal of the protein of interest when it is being viewed under a microscope, but can also increase the chance of off-target labeling. Similarly, secondary antibodies must always be created in a different animal than the primary antibody to cross-reactivity: labeling cells where no antigen is present. Finally, when performing the IHC protocol, scientists must carefully dilute their antibodies in solution and wash away any excess between steps to keep the reactive elements limited to the cells where the antigen genuinely exits.
The elegance and broad applications of IHC have helped make this technique a staple for biologists of many fields. Neuroscientists in particular have found it useful to tease apart the many layers of cells and their various subtypes in the brain. Next time you see a beautiful, multi-colored image of the brain produced by a Max Planck lab, check out their website to see if IHC was used to help produce it.