engleski

Detergent-induced membrane redistribution of molecules with lipid anchor as a source of common artifacts in fixed tissue sections. Examples from brain development and regeneration.

Immunohistochemical analysis is a common basic method that is a prerequisite for following functional studies and in vivo imaging. However, antibodies are large molecules that can not cross intact membrane, thus cells have to be treated to become permeable. Paraffin embedded sections are permeabilized during deparaffinization and additional permeabilization step is usually not needed. Various other types of sections (fresh or fixed, frozen or not frozen) and even cell cultures, require initial step of cell permeabilization, prior to immunolocalization of molecules in cytoplasm, organelles or compacted membranes, like myelin. Permeabilization is efficiently performed by organic solvents and detergents, or less common and less successive, by freezing and thawing. Each of these methods solubilize membrane lipids, but it is not well known to which extent this happens in fixed tissue and how it affects original distribution of membrane molecules. This is especially the case with molecules that reside in lipid rafts and are considered detergent-insoluble. Using different immunochemical procedures we studied a set of molecules involved in the development and regeneration of CNS that are either integral membrane proteins (MAG, p75NTR, PSA-NCAM), or only attached to the outer leaflet of the membrane through lipid anchor (NogoR, gangliosides, PSA-NCAM, Omgp). Different detergents and detergent concentrations, and varying time and temperature, were found to affect different types of molecules differently, leading to apparently different immunochemical localization. It seems that the use of detergents compromises immunochemical analysis of glycolipids and GPI-anchored proteins and their natural distribution has to be determined using methods in which detergents are not being used.

brain; immunohistochemistry; axonal regeneration

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