Saturday, March 27, 2010
So how do we find out what’s happening in the brain? To visual the neurodegeneration that occurs in Alzheimer’s disease, we use a process called immunohistochemstry. A technology that allows us to detect the expression of specific proteins in the brain using antibodies that are manufactured to seek out the unique shape of each individual protein.
The first step is to fix the brain tissue with paraformaldehyde. This serves three purposes. First, it freezes the brain in time, so affectively we get a snapshot of the state the brain was in at the time of fixation, secondly, it gives fantastic integrity to the tissue so it doesn’t fall apart during processing, and thirdly, it forms large formaldehyde bonds, cross-linking the proteins so the antibody can recognize the exact shape of each protein. The next step is to section the brain, the sections used for these images is from 40micron brain sections (that’s 1/25th of a millimeter). To achieve this we freeze the brain to -20C then slice it using an extremely sharp blade attached to a machine called a microtome. From a single mouse brain you end up with around 300 sections, which have to be reassembled back into order on glass slides (see
Queensland Brain Institute post by Melody).
The sections next have to be prepared to accept the antibody. All cells, including brain cells have a lipid (fat) cell wall that the antibody needs to penetrate. To do this we soak the brains ethanol, which breaks down the integrity of the cell wall allowing the antibody to move inside the cell. The specificity of the antibody antigen relationship is then further enhanced by soaking the brains in animal plasma and hydrogen peroxide. This prevents the antibodies from binding to non-specific antigens and endogenous peroxidases. The brain tissue is then incubated with an antibody that recognizes your specific protein of interest. This process usually takes around 48 hours. The antibodies are used at incredibly low concentrations, around 1 part antibody to 100,000 parts of buffer (see picture of antibody preparation in Green glow post by Melody).
Binding secondary antibodies that seek out the primary antibodies then further enhances the signal. If the signal is weak then occasionally a tertiary antibody is used to further enhance the signal. These antibodies have a fluorescent tag attached to them.
The sections are then mounted onto a microscope slide. It’s a bit like doing fine art and jigsaw puzzles, as the brain has to be put back in correct order by firstly sorting out the sections into their correct order then gently manipulating a very fragile 40-micron slice of brain tissue onto a piece of glass. Depending on the amount of tissue this process can take 3 or 4 days to complete. The tissue is then coverslipped in a highly alkaline viscous buffer solution so they can finally be viewed on the microscope.
The microscopes used to visualize the fluorescent signal are an amazing piece of technology, costing as mush as a house. Using lasers to excite the fluorophores the highly powerful CCD digital cameras capture the emission wavelength emitted by the fluorescent tag. Any changes in the expression of a protein of interested manipulated by the experimental design can then be analyzed.