Immuno-localization of Fas and FasL in rat hepatic endothelial cells: influence of different fixation protocols

Immunocytochemistry has been widely used to localize molecules involved in apoptosis. In this short report, we describe with the aid of confocal laser scanning microscopy the immunolocalization of Fas and FasL on liver sinusoidal endothelial cells, and show how the localization of these two molecules differ when the cells are fixed with different fixation protocols. Methanol fixation shows diffuse staining of Fas and FasL in the cytoplasm, as well as in the nucleus. In contrast, paraformaldehyde fixation reveals the presence of Fas and FasL polarized at one side of the cell and only in the cytoplasm. After fixation with a combination of paraformaldehyde and glutaraldehyde the polarization is still present although the fluorescence is concentrated and located as bright dots in the cytoplasm. In conclusion, paraformaldehyde preserves the (nuclear) membrane-associated structures better then methanol and results in a more accurate localization of Fas and FasL. Understanding the different outcome of these common used fixation protocols will assist investigators to select the most suitable method for visualizing membrane-bound Fas and FasL.