Improved procedures for immunoferritin labeling of ultrathin frozen sections

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.

[1]  K. Tokuyasu Membranes as observed in frozen sections. , 1976, Journal of ultrastructure research.

[2]  J. Kyte Immunoferritin determination of the distribution of (Na+ + K+) ATPase over the plasma membranes of renal convoluted tubules. I. Distal segment , 1976, The Journal of cell biology.

[3]  S. Singer,et al.  A disulfide-bridge bifunctional imidoester as a reversible cross-linking reagent. , 1975, Biochemical and biophysical research communications.

[4]  B. Olsen,et al.  Two improved methods for preparing ferritin-protein conjugates for electron microscopy , 1975, The Journal of cell biology.

[5]  A. Hand,et al.  TISSUE FIXATION WITH DIIMIDOESTERS AS AN ALTERNATIVE TO ALDEHYDES I. COMPARISON OF CROSS-LINKING AND ULTRASTRUCTURE OBTAINED WITH DIMETHYLSUBERIMIDATE AND GLUTARALDEHYDE , 1974, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[6]  S. Singer,et al.  Immunoferritin localization of intracellular antigens: the use of ultracryotomy to obtain ultrathin sections suitable for direct immunoferritin staining. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[7]  K. Tokuyasu A TECHNIQUE FOR ULTRACRYOTOMY OF CELL SUSPENSIONS AND TISSUES , 1973, The Journal of cell biology.

[8]  G. Nicolson ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES , 1973, The Journal of cell biology.

[9]  J. Kraehenbuhl,et al.  Solid-phase conjugation of ferritin to Fab-fragments of immunoglobulin G for use in antigen localization on thin sections. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[10]  S. Singer,et al.  ALTERATION OF THE CONFORMATION OF PROTEINS IN RED BLOOD CELL MEMBRANES AND IN SOLUTION BY FIXATIVES USED IN ELECTRON MICROSCOPY , 1968, The Journal of cell biology.

[11]  S. Singer,et al.  Bifunctional imidoesters as cross-linking reagents. , 1966, Biochemical and biophysical research communications.

[12]  S. J. Singer,et al.  THE PROPERTIES OF SPECIFIC STAINS FOR ELECTRON MICROSCOPY PREPARED BY THE CONJUGATION OF ANTIBODY MOLECULES WITH FERRITIN , 1961, The Journal of biophysical and biochemical cytology.

[13]  S. Singer,et al.  A general method for the specific staining of intracellular antigens with ferritin-antibody conjugates. , 1970, Proceedings of the National Academy of Sciences of the United States of America.