Hello, everyone. I would like to determine the fiber type of each skeletal muscle fiber based on its SDH and ATPase activity.I used the dry ice-hexane method to freeze freshly isolated skeletal muscle because the liquid nitrogen freezing method produced bubble artifacts. Although these methods were used to evaluate by enzyme activity, there is unavoidable bubble artifact.
If it is difficult, I will prepare frozen sections after saturation with normal sucrose and use an antibody-based staining method for each muscle fiber.
If you have any knowledge, I would appreciate it very much.
Thank you in advance.
The most common procedure to avoid artifacts is to freeze muscle in isopentane cooled in liquid nitrogen. In other words, the isopentane is added to a small container, and this container is put in a larger container containing liquid nitrogen. When the isopentane is cooled to its freezing temperature -- it starts to becomes a white solid -- then it is ready.
Thank so much Dr. Jeffrey J Brault. Very helpful. From your advice, It seems from the method I tried that the temperature at which the sample was frozen was still too high, or was affected by the liquid nitrogen itself.I will try it. Thank you so much. I appreciate it.
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