Summary
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 a queous 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° C with silver proteinate (SP). The ninhydrin-dimethyl sulfoxide-thiocarbohydrazidesilver 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.
Similar content being viewed by others
References
Dalen JPR van, Duijn P van (1971) Quantitative aspects of the ninhydrin-Schiff reaction on amino cellulose films and tissue sections. Histochemie 26:180–189
Gander ES Moppert JB (1969) Der Einfluß von Dimethyl Sulfoxide auf die Permeabilität der Lysosomenmembran bei quantitativer und qualitativer Darstellung der sauren Phosphotase. Histochemie 20:211–214
Glenner GG (1963) A re-evaluation of the ninhydrin-Schiff reaction. J Histochem Cytochem 11:285–286
Gomori G (1952) Histochemistry. University of Chicago Press, Chicago, p 113
Kasten FH (1963) Some comments on a recent criticism of the ninhydrin-Schiff reaction. J Histochem Cytochem 10:769–770
Kirschenbaum DM (1965) Modification of the technicon “N” methodology for automatic amino acid analysis. Anal Biochem 12:189–198
Kornfeld S (1987) Trafficking of lysosomal enzymes. FASEB J 1:462–468
Lo HK, Malinin TI, Malinin GI (1987) A modified periodic acidthiocarbohydrazide-silver proteinate staining sequence for enhanced contrast and resolution of glycogen deposition by transmission electron microscopy. J Histochem Cytochem 35:393–399
Malinin GI (1976a) Dimethyl sulfoxide-lead tetraacetate method for histochemical oxidation of polysacharides. J Histochem Cytochem 24:443–447
Malinin GI (1976b) Selective histochemical localization of polysaccharides in tissue sections oxidized by acetic anhydride in dimethyl sulfoxide. J Histochem Cytochem 24:993–999
Malinin GI (1977) Oxidation of tissue polysaccharides by periodic acid in dimethyl sulfoxide and its anhydrous and aqueous mixtures. J Histochem Cytochem 25:188–192
Malinin GI, Lo HK, Malinin TI (1987) Electron microscopy of hepatic cells oxidatively deaminated with ninhydrin in dimethyl sulfoxide. J Histochem Cytochem 35:1920 (ABS)
Misch DW, Misch MS (1969) Dimethyl sulfoxide: activation of lysosomes in vitro. Proc Natl Acad Sci USA 58:2462–2467
Misch DW, Misch MS (1969) Reversible activation of lysosomes by dimethyl sulfoxide. Nature 221:862–863
Misch DW, Misch MS (1973) Histochemical activation of rat-liver lysosomes by dimethyl sulfoxide. Histochemistry 37:131–140
Moore S (1968) Amino acid analysis: aqueous dimethyl sulfoxide as solvent for ninhdrin reaction. J Biol Chem 243:6281–6283
Moore S, Stein WH (1948) Photometric ninhydrin method for use in the chromatography of amino acids. J Biol Chem 176:367–388
Morii S, Takigami S, Kaneda Y, Shikata N (1982) Ultracytochemical analysis of cytoplasmic lipids by enzymatic digestive methods. Acta Histochem Cytochem 15:185–197
Neiss WF (1988) Enhancement of the periodic acid-Schiff (PAS) and periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) reaction in LR White sections. Histochemistry 88:603–612
Newman GR (1987) Use of abuse of LR White. Histochem J 19:118–120
Reynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208–213
Ruhemann S (1911) Triketohydrindene hydrate Part 3. Its relation to alloxan. J Chem Soc 99:792–800
Sanyal S (1970) Changes of lysosomal enzymes during hereditary degeneration and histogenesis of retina in mice. Histochemistry 23:207–219
Schrével J, Gros D, Monsigny M (1981) Cytochemistry of cell glycoconjugates. Prog Histochem Cytochem 14:1–269
Seligman AM, Hanker JS, Wasserkrug H, Dmochowski H, Katzoff H (1965) Histochemical demonstration of some oxidized macromolecules with thiocarbohydrazide (TCH) or thiosemicarbazide (TSC) and osmium tetroxide. J Histochem Cytochem 13:629–639
Szmant HH (1971) Chemistry of DMSO. In: Jacob SW, Rosenbaum EE, Wood DC (eds) Dimethyl sulfoxide. Marcel Dekker, New York, pp 1–97
Yasuma A, Ichikawa T (1953) Ninhydrin-Schiff and alloxan-Schiff staining. J Lab Clin Med 41:296–299
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Malinin, G.I., Lo, H.K. & Malinin, T.I. Ultrastructural cytochemistry of hepatic lysosomes and their protein components is selectively revealed by the ninhydrin-dimethyl sulfoxide-thiocarbohydrazide-silver proteinate reaction. Histochemistry 90, 339–345 (1989). https://doi.org/10.1007/BF00508310
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00508310