Components of muscle
The myology laboratory has over the years increased its capacity for self sustained tissue processing, sectioning and imaging. We are very fortunate to possess our own cryostat (refrigerated microtome), a fluorescence microscope, a bright field microscope, and a stereo microscope, all fitted with high resolution cameras and acquisition software.
There are a number of advantages of using immunofluorescence over the conventional bright field method. First, multiple proteins can be labeled with specific antibodies and visualised independently, or in combination with each other. The sequence of photographs below illustrates this approach using skeletal muscle from the cheetah. A muscle section was probed for type I (blue), type IIA (green), and type IIX (red) fibres and photographed separately. The photos were merged into one final picture to reveal the location of these three fibre types. It is from this picture that muscle fibre type is determined.
Using advanced imaging software, we are also able to determine the size of each fibre (cross-sectional area), and calculate an average for each fibre type. Fluorescent immunohistochemistry is only limited to the availability of the primary antibodies. We currently have over 20 muscle structural primary antibodies that can be used for research, from dystrophin, the various myosin heavy chain isoforms, myosin light chains, troponin, and many more.
Bright field and stereo imaging
We also routinely perform a number of metabolic stains, including the NADH and succinate dehydrogenase stain to show the location and density of mitochondria in the various fibre types. These stains target mitochondria: the darker the intensity, the more mitochondria present in the muscle.
Our newest addition to the lab is a Zeiss Stemi 305 stereo microscope. It expands the lab’s capability to acquire not only pictures from muscle, but from other materials such as plant material or synthetics.
Gel electrophoresis and Western blotting
Gel electrophoresis is a protein separating technique. Western blotting is an extension of electrophoresis in order to visualise the separated proteins. It entails the transfer of the separated proteins to a solid material that can be probed with antibodies to visualise only the protein of interest. This is probably the most used technique in the world. We also use other visualisation techniques, like Coomasie and silver staining.
Western blotting is much more sensitive than fluorescent IHC, partly because of the separation and concentration of the target protein from the rest of the cellular proteins. Another advantage is that the sample preparation is not dependent on the integrity of the tissue cells. In other words: many tissue samples that were stored for a very long time or that accidentally defrosted (freezer failure), are not suitable for IHC due to cellular membrane disruptions. However, this tissue can still be used for electrophoresis, Western blotting and to some extent the metabolic enzyme kinetics.