A “non-classical” and reliable method for transmission immunoelectron microscopy.

Sample preparation for transmission immunoelectron microscopy techniques is pivotal to preserve cell architecture and to maintain antigen epitopes. We propose a “non-classical” method in order to obtain the above-mentioned conditions. In a pilot series of experiments, we found a mixture of 2.0% paraformaldehyde/0.1% glutaraldehyde in 0.1 M PBS (pH 7.4) to be an optimal fixing solution. In this way, there was a minimal cover of antigen epitopes. To maintain a good cell morphology and a stronger contrast between cellular compartments we also performed a post fixation in 1% OsO4 for 1h at 4°C and an embedding in epoxy resin. We did not apply the routine method of embedding specimens in acrylic resins, since such a procedure does preserve antigens but also impairs ultrastructural integrity. In our methodological experiments the use of epoxy resin was essential to preserve subcellular structures and to combine an optimal cell trim with an acceptable integrity of antigen epitopes. In this paper we report data regarding the application of this immunoelectron microscopy technique to animal cells and tissue.