Immunogold-labelling

The aim of immunocytochemistry is to localize atigens by labelling them with specific antibodies. Thus the fixation procedure is chosen to preserve antigenicity, even if it is not good for ultrastructure. The reactivity and stability of each antigen differs so widely from one antigen to another that no one method of fixation can be guaranteed to work for all EM immunocytochemistry experiments. Few antigens survive the routine fixation and embedding procedures which utilize osmium tetroxide or uranyl acetate as additional fixatives. Instead, we tend to use a more gentle aldehyde fixation, combined with low temperature embedding. This has been shown to help preserve the antigenicity of the sample but, to some extent, this is at the expense of high quality ultrastructure.

Once the material is suitably embedded, thin sections are made and picked up on gold grids (as many of the reagents used in immunolabelling procedures would etch copper). The sections can be immunolabelled by floating the grids on the surface of reagent droplets and washing in between by floating on the surface of a larger volume of buffer or water. To see the labelling in the electron microscope, we must use gold-conjugated secondary antibodies to provide areas of high electron density. These can be purchased in various sizes (e.g. 10nm and 20nm particles) to allow for double labelling experiments. Gold antibodies can also be used in conjunction with silver enhancement for the light microscope. Very small gold particles (1-3nm) can be used, together with silver enhancement, for the TEM and if they are conjugated to a Fab antibody fragment, it has been found to giver better penetration of the probe due to it's small size, and higher labelling density. For further information, see http://www.nanoprobes.com.