Distribution of manganese superoxide dismutase in rat stomach: application of Triton X-100 and suppression of endogenous streptavidin binding activity.
The distribution of rat manganese superoxide dismutase (Mn-SOD) was immunohistochemically investigated in the rat stomach with a specific polyclonal antibody and a labeled streptavidin-biotin immunoglobulin detection system in cryosections. Parietal cells in the stomach were intensely stained, whereas the other epithelial cells in the gastric gland and pit exhibited only slight staining. Rapid-freezing and freeze-substitution immunoelectron microscopy revealed that Mn-SOD in parietal cells was mainly localized in mitochondria. Therefore, the large amount of Mn-SOD in parietal cells is due to the abundant mitochondria, in which Mn-SOD is considered to play important roles in protecting the ion pump and the cell itself from superoxide insult. Application of Triton X-100, cryosectioning, and the streptavidin-biotin system are needed to distinctly visualize Mn-SOD with our antibody. Treatment of the cryosections with Triton X-100 enhanced not only the immunoreactivity but also the false-positive staining, which showed a similar distribution pattern to that of Mn-SOD and thus made it difficult to determine the localization. The most plausible cause of the false-positive staining is thought to be endogenous biotin in the stomach, which survives paraformaldehyde fixation and is revealed by Triton X-100 treatment. Suppression of the endogenous streptavidin binding activity is important when cryosections, the streptavidin-biotin system, and Triton X-100 are employed.
Satoh
S
S
Department of Anatomy, Sapporo Medical College, Japan.
Tatsumi
H
H
Suzuki
K
K
Taniguchi
N
N
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D005189:False Positive Reactions; D005753:Gastric Mucosa / Q000201:enzymology*; D007124:Immunoenzyme Techniques; D008297:Male; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000201:enzymology; D017830:Octoxynol; D010295:Parietal Cells, Gastric / Q000201:enzymology* / Q000648:ultrastructure; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013270:Stomach / Q000201:enzymology*; D019809:Streptavidin; D013482:Superoxide Dismutase / Q000276:immunology / Q000378:metabolism*
D001426:Bacterial Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D019809:Streptavidin; D013482:Superoxide Dismutase