Ultrastructural cytochemistry of hepatic lysosomes and their protein components is selectively revealed by the ninhydrin-dimethyl sulfoxide-thiocarbohydrazide-silver proteinate reaction.
Proteins in lysosomal membranes, lysosomes and within the transtubular network are readily accessible for electron microscopic analysis by a new three-step method. Oxidative deamination of tissue-bound amino acids by ninhydrin in aqueous dimethyl sulfoxide and the concomitant formation of corresponding carbonyl groups comprise the first step. The addition reaction of thiocarbohydrazide to tissue-bound carbonyl groups comprises the second step, while the reduction of silver proteinate by tissue-bound thiocarbohydrazones is the final step of this sequential method. Glutaraldehyde-fixed and osmified ultrathin sections of rat liver embedded in LR White were oxidatively deaminated for 24 h by 1% w/v ninhydrin in aqueous 75% v/v dimethyl sulfoxide (DMSO). They were then incubated for 40 min in aqueous 1% w/v thiocarbohydrazide (TCH) and stained for 30 min at 50 degrees C with silver proteinate (SP). The ninhydrin-dimethyl sulfoxide-thiocarbohydrazide-silver proteinate (N-DMSO-TCH-SP) reaction proved to be chemically specific and highly selective for ultrastructural resolution of the internal structure of lysosomes and their protein components. We conclude that the N-DMSO-TCH-SP reaction is the method of choice for cytochemical elucidation of the protein ultrastructure of lysosomes and their enzymatic aggregates.
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Department of Physics, Georgetown University, Washington, DC 20057.
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D000818:Animals; D004121:Dimethyl Sulfoxide; D006834:Hydrazines; D007202:Indicators and Reagents; D008099:Liver; D008247:Lysosomes / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods*; D009555:Ninhydrin; D051381:Rats; D011919:Rats, Inbred Strains; D012836:Silver Proteins
D006834:Hydrazines; D007202:Indicators and Reagents; D012836:Silver Proteins; C011368:thiocarbohydrazide; D009555:Ninhydrin; D004121:Dimethyl Sulfoxide