Post-embedding immuno-electron microscopy with rapid freezing and freeze-substitution techniques for the localization...

Dehydration of specimens with ethanol or acetone makes it impossible to detect manganese superoxide dismutase (Mn-SOD) by immunohistochemistry. To circumvent obstacles and demonstrate localization by post-embedding immuno-electron microscopy, a rapid freezing and freeze-substitution technique was employed using Lowicryl K4M embedding medium. This was effective enough to allow the specific observation of immunogold particles for Mn-SOD on the mitochondria of cardiac muscle cells and WI-38 cells (human normal fetal lung diploid cells). This method preserved the antigen-antibody binding activity of Mn-SOD even after dehydration. Therefore, rapid freezing and freeze-substitution is useful for post-embedding immuno-electron microscopy of Mn-SOD and can further be employed for other antigens previously difficult to detect by conventional methods.