Improved pre-embedded immuno-electron microscopy procedures to preserve myelin integrity in mammalian central nervous tissue

Preparation of specimens for pre-embedded immuno-electron microscopic analysis of CNS tissue is challenging. Conventional methods frequently result in significant damage to myelin integrity, ultimately confounding accurate analysis of myelin quality and assessment of changes in the number of lamellae. We have developed and optimized a protocol for maintaining good myelin morphology in CNS tissue while allowing for detect of antigens of interest with immunocytochemistry. We demonstrate that the presence of potassium ferrocyanide during lipid fixation is critical for obtaining optimal myelin ultrastructure. Further, our data illustrate the importance of rapid and efficient fixation for preservation of myelin integrity. Using this optimized technique, studies investigating potential efficacy of remyelination via administration of specific treatments can accurately distinguish between degenerating myelin after CNS injury/disease and new myelin (loose or compact) formed as a result of the intervention strategy.