Immunocytochemistry is a method to localize a special constituent with labelled antibodies. In the first step, the primary antibody binds specific to the target antigen. In the second step, a gold-labelled secondary antibody is applied which binds to the primary antibody. The gold particles are well visible in the electron microscope and give information about the distribution of the antigen in the thin slice of the specimen. Using gold-labeled secondary antibodies with different sizes of the gold particles allows the visualization of different antigens at the same time.
Prior to immunocytochemistry the specimens need to be prepared. The most important fact is to make sure that the antigenic sites of the antigen are kept available to the antibody applied.
The LEM offers labelling of (a) cryosections and (b) resin section.
(a) Immunolabelling of ultrathin cryosections
The specimens are fixed with paraformaldehyde and glutaraldehyde. In a following step cryoprotective agents are applied to the specimens prior freezing in liquid nitrogen. After ultrathin sectioning of the frozen specimens the sections are mounted on a coated grid.
The labelling starts with blocking the aldehydes and unspecific binding sites. Incubation with the primary antibody and the secondary antibody with washing steps in between follows. Finally, the sections are stabilized with methyl cellulose and simultaneously stained with uranyl acetate.
(b) Immunolabelling of resin sections
For the so-called postembedding method specimens are fixed, dehydrated and infiltrated with resin. After sectioning using an ultramicrotome the sections are mounted on a grid and immersed in different buffers to block the aldehydes and unspecific binding sites. Incubation with the primary antibody and the secondary antibody with washing steps in between follows. Finally, sections are stained with uranyl acetate (and lead citrate) and can be examined with the transmission electron microscope.