Author Of 1 Presentation
P0970 - Immunoelectron microscopy for ultrastructural studies of oligodendrocyte progenitors (ID 1778)
Abstract
Background
Oligodendrocytes and its progenitors play a crucial role in MS, not only in the pathogenesis but also in remyelination. To asset the role of these cells the first step is their identification either in human tissue or animal models, for this purpose, specific protein labeling is the most broadly used technique. Marking specific proteins allows us to determine the stage of maturation of each oligodendrocyte in every area orf the brain. On the other hand, electron microscopy is one of the most useful tools to describe the fine morphology, ultrastructure and contacts formed by any cell in the brain. Furthermore, electron microscopy is considered to be the gold satandard to determine myelination and remyelination. The combination of both techniques allows the subcellular localization of proteins and the description of the morphology of specific cells. However, the most commonly used oligodendrocyte progenitor markers are challenging to observe through transmission electron microscopy while maintaining the ultrastructural characteristics of the cell. Hereby, we present a novel method that uses tyramide signal amplification (TSA) to identify oligodendrocyte progenitors.
Objectives
To ultrastructurally identify oligodendrocyte progenitors through immunnoelectron microscopy in human and murine tissue.
Methods
To take advantage of the signal amplification provided by TSA, we have developed a methodology that significantly reduces the time needed for primary antibody incubation in standard immunogold protocols, and only requires ~25% of the antibody concentration that is normally used. Furthermore, this technique allows the precise visualization of some antigens that were not labelable by standard immunoelectron microscopy such as mouse NG2; human and mouse PDGFRa; or antigens whose label is not strong enough to be distinguished from background noise, such as BCAS1. Our approach is based on TSA-biotin labeling together with standard immunoelectron microscopy.
Results
We have achieved to visualize antigens that were previousy undetectable through conventional immunogold methods such as PDGFRα, NG2 and BCAS1 not only in murine but in human tissue. Our results show that PDGFRα is a very specific marker for oligodendrocyte progenitor cells and which labeling is specific to some membrane domains in human subcortical white matter. NG2 which is considered to be a marker of glial/oligodendrocyte precursors, is distributed not only in the membrane, but also in the cytoplasm and associated to intermediate filaments in human subcortical white matter. Finally the novel marker for myelinating preoligodendrocytes BCAS1 labels cells with prominent expansions which are associated to myelin in the same brain area.
Conclusions
The specificity and sensitivity of TSA to label these antigens shows that this technique is valid for futher ultrastructural studies on the basic biology of oligodendrocytes and myelination.