Antigen preservation tests for immunocytochemical detection of cytoskeletal proteins: influence of aldehyde fixatives.
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.
Riederer
B M
BM
Department of Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D000941:Antigens / Q000276:immunology*; D001369:Axons / Q000378:metabolism / Q000648:ultrastructure; D001921:Brain / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000276:immunology / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D005404:Fixatives / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods*; D007381:Intermediate Filament Proteins / Q000276:immunology / Q000378:metabolism; D007382:Intermediate Filaments / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016900:Neurofilament Proteins; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats
D000447:Aldehydes; D000941:Antigens; D003598:Cytoskeletal Proteins; D005404:Fixatives; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins