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Antigen preservation tests for immunocytochemical detection of cytoskeletal proteins: influence of aldehyde fixatives.
Journal of Histochemistry and Cytochemistry
The effects of aldehyde fixatives on immunochemical detection of cytoskeletal proteins were demonstrated by applying several quantitative assays to evaluate antigen conservation. Immunologically detectable brain spectrin (240/235) was measured by dot-immunobinding and quantitative immunodot assay using a polyclonal antibody. Paraformaldehyde fixation led to a 43-66% reduction in brain spectrin (240/235) immunodetection, and increasing glutaraldehyde concentrations decreased the immunological detection even more. Quantitative cryosection immunoassay and immunocytochemical localization confirmed the aldehyde sensitivity of brain spectrin (240/235). Brain spectrin (240/235) immunoreactivity decreased with increasing protein crosslinking and was dependent on glutaraldehyde concentration and post-fixation period. The assays were also used to test for conservation of antigenicity of neurofilament proteins by two monoclonal antibodies. Neurofilament detection was abolished in brain tissue after aldehyde fixation. The described methods allow screening within 24 hr of many fixation conditions by use of purified proteins as well as brain tissue samples, and allow an estimate of fixative influence on the conservation of protein antigenicity.
Aldehydes/diagnostic use, Aldehydes/pharmacology, Animals, Antigens/immunology, Axons/metabolism, Axons/ultrastructure, Brain/cytology, Brain/metabolism, Cytoskeletal Proteins/immunology, Cytoskeletal Proteins/metabolism, Cytoskeleton/drug effects, Cytoskeleton/metabolism, Fixatives/diagnostic use, Fixatives/pharmacology, Immunohistochemistry/methods, Intermediate Filament Proteins/immunology, Intermediate Filament Proteins/metabolism, Intermediate Filaments/drug effects, Intermediate Filaments/metabolism, Mice, Neurofilament Proteins, Neurons/metabolism, Neurons/ultrastructure, Rats
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