Post-embedding immuno-electron microscopy with rapid freezing and freeze-substitution techniques for the localization of manganese superoxide dismutase.
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.
Tatsumi
H
H
Department of Anatomy, Sapporo Medical College, Japan.
Suzuki
K
K
Senda
T
T
Satoh
S
S
Fujita
H
H
Taniguchi
N
N
D000180:Acrylic Resins; D000818:Animals; D002460:Cell Line; D005615:Freezing; D005629:Frozen Sections; D005753:Gastric Mucosa / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000201:enzymology / Q000648:ultrastructure; D008929:Mitochondria, Heart / Q000201:enzymology / Q000648:ultrastructure; D051381:Rats; D013482:Superoxide Dismutase / Q000032:analysis*; D016610:Tissue Embedding
D000180:Acrylic Resins; C037883:Lowicryl 4KM resin; D013482:Superoxide Dismutase