Abstract
In employing fixed frozen ultrathin sections as substrates for immunoferritin labeling of intracellular antigens, we have found that conventional glutaraldehyde fixation sometimes permits very little specific staining of the sections, either because it inactivates certain protein antigens, or because it renders them inaccessible to the antibody stains. We have developed several fixation procedures that are chemically milder and allow a uniform but less extensive cross- linking of the specimen. With these procedures and precautions in the handling of the more fragile frozen sections, excellent structural preservation and specific immunoferritin labeling has been achieved with several systems.
Full Text
The Full Text of this article is available as a PDF (5.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Dutton A., Adams M., Singer S. J. Bifunctional imidoesters as cross-linking reagents. Biochem Biophys Res Commun. 1966 Jun 13;23(5):730–739. doi: 10.1016/0006-291x(66)90462-1. [DOI] [PubMed] [Google Scholar]
- Hassell J., Hand A. R. Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of cross-linking and ultrastructure obtained with dimethylsuberimidate and glutaraldehyde. J Histochem Cytochem. 1974 Apr;22(4):223–229. doi: 10.1177/22.4.223. [DOI] [PubMed] [Google Scholar]
- Kishida Y., Olsen B. R., Berg R. A., Prockop D. J. Two improved methods for preparing ferritin-protein conjugates for electron microscopy. J Cell Biol. 1975 Feb;64(2):331–339. doi: 10.1083/jcb.64.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lenard J., Singer S. J. Alteration of the conformation of proteins in red blood cell membranes and in solution by fixatives used in electron microscopy. J Cell Biol. 1968 Apr;37(1):117–121. doi: 10.1083/jcb.37.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nicolson G. L., Painter R. G. Anionic sites of human erythrocyte membranes. II. Antispectrin-induced transmembrane aggregation of the binding sites for positively charged colloidal particles. J Cell Biol. 1973 Nov;59(2 Pt 1):395–406. doi: 10.1083/jcb.59.2.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Painter R. G., Tokuyasu K. T., Singer S. J. Immunoferritin localization of intracellular antigens: the use of ultracryotomy to obtain ultrathin sections suitable for direct immunoferritin staining. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1649–1653. doi: 10.1073/pnas.70.6.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruoho A., Bartlett P. A., Dutton A., Singer S. J. A disulfide-bridge bifunctional imidoester as a reversible cross-linking reagent. Biochem Biophys Res Commun. 1975 Mar 17;63(2):417–423. doi: 10.1016/0006-291x(75)90704-4. [DOI] [PubMed] [Google Scholar]
- Tokuyasu K. T. A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol. 1973 May;57(2):551–565. doi: 10.1083/jcb.57.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tokuyasu K. T. Membranes as observed in frozen sections. J Ultrastruct Res. 1976 May;55(2):281–287. doi: 10.1016/s0022-5320(76)80073-1. [DOI] [PubMed] [Google Scholar]