Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1963 Apr 1;17(1):19–58. doi: 10.1083/jcb.17.1.19

CYTOCHEMISTRY AND ELECTRON MICROSCOPY

The Preservation of Cellular Ultrastructure and Enzymatic Activity by Aldehyde Fixation

David D Sabatini 1, Klaus Bensch 1, Russell J Barrnett 1
PMCID: PMC2106262  PMID: 13975866

Abstract

The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.

Full Text

The Full Text of this article is available as a PDF (5.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BARRNETT R. J., PALADE G. E. Applications of histochemistry to electron microscopy. J Histochem Cytochem. 1958 Jan;6(1):1–12. doi: 10.1177/6.1.1. [DOI] [PubMed] [Google Scholar]
  2. BARRNETT R. J., PALADE G. E. Enzymatic activity in the M band. J Biophys Biochem Cytol. 1959 Oct;6:163–170. doi: 10.1083/jcb.6.2.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BARRNETT R. J. The fine structural localization of acetylcholinesterase at the myoneural junction. J Cell Biol. 1962 Feb;12:247–262. doi: 10.1083/jcb.12.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BEAUFAY H., DE DUVE C., HOLT S. J., UNDERHAY E. Intracellular localization of esterase in rat liver. J Biophys Biochem Cytol. 1956 Sep 25;2(5):635–637. doi: 10.1083/jcb.2.5.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. BRANDES D., ZETTERQVIST H., SHELDON H. Histochemical techniques for electron microscopy: alkaline phosphatase. Nature. 1956 Feb 25;177(4504):382–383. doi: 10.1038/177382a0. [DOI] [PubMed] [Google Scholar]
  6. Bensch K. G., King D. W. Incorporation of Heterologous Deoxyribonucleic Acid into Mammalian Cells. Science. 1961 Feb 10;133(3450):381–382. doi: 10.1126/science.133.3450.381. [DOI] [PubMed] [Google Scholar]
  7. CAULFIELD J. B. Effects of varying the vehicle for OsO4 in tissue fixation. J Biophys Biochem Cytol. 1957 Sep 25;3(5):827–830. doi: 10.1083/jcb.3.5.827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. ESSNER E., NOVIKOFF A. B., MASEK B. Adenosinetriphosphatase and 5-nucleotidase activities in the plasma membrane of liver cells as revealed by electron microscopy. J Biophys Biochem Cytol. 1958 Nov 25;4(6):711–716. doi: 10.1083/jcb.4.6.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. GOMORI G. Alkaline phosphatase of cell nuclei. J Lab Clin Med. 1951 Apr;37(4):526–531. [PubMed] [Google Scholar]
  10. HOLT S. J., HICKS R. M. Studies on formalin fixation for electron microscopy and cytochemical staining purposes. J Biophys Biochem Cytol. 1961 Oct;11:31–45. doi: 10.1083/jcb.11.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. HUXLEY H. E., ZUBAY G. Preferential staining of nucleic acid-containing structures for electron microscopy. J Biophys Biochem Cytol. 1961 Nov;11:273–296. doi: 10.1083/jcb.11.2.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. KARRER H. E. Electron microscope observations on chick embryo liver. Glycogen, bile canaliculi, inclusion bodies and hematopoiesis. J Ultrastruct Res. 1961 Apr;5:116–141. doi: 10.1016/s0022-5320(61)90009-0. [DOI] [PubMed] [Google Scholar]
  13. LEDUC E. H., BERNHARD W. Ultrastructural cytochemistry. Enzyme and acid hydrolysis of nucleic acids and protein. J Biophys Biochem Cytol. 1961 Jul;10:437–455. doi: 10.1083/jcb.10.3.437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. NACHLAS M. M., TSOU K. C., DE SOUZA E., CHENG C. S., SELIGMAN A. M. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J Histochem Cytochem. 1957 Jul;5(4):420–436. doi: 10.1177/5.4.420. [DOI] [PubMed] [Google Scholar]
  16. NAIDOO D., PRATT O. E. The development of adenosine 5'-phosphatase activity with the maturation of the rat cerebral cortex. Enzymologia. 1954 Mar 15;16(5):298–304. [PubMed] [Google Scholar]
  17. NOVIKOFF A. B., GOLDFISCHER S. Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc Natl Acad Sci U S A. 1961 Jun 15;47:802–810. doi: 10.1073/pnas.47.6.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. PALADE G. E. A study of fixation for electron microscopy. J Exp Med. 1952 Mar;95(3):285–298. doi: 10.1084/jem.95.3.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. PALADE G. E., SIEKEVITZ P. Pancreatic microsomes; an integrated morphological and biochemical study. J Biophys Biochem Cytol. 1956 Nov 25;2(6):671–690. doi: 10.1083/jcb.2.6.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. REVEL J. P., NAPOLITANO L., FAWCETT D. W. Identification of glycogen in electron micrographs of thin tissue sections. J Biophys Biochem Cytol. 1960 Dec;8:575–589. doi: 10.1083/jcb.8.3.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. SCARPELLI D. G., HESS R., PEARSE A. G. The cytochemical localization of oxidative enzymes. I. Diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase. J Biophys Biochem Cytol. 1958 Nov 25;4(6):747–752. doi: 10.1083/jcb.4.6.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. SENO S., YOSHIZAWA K. Electron microscope observations on frozen-dried cells. J Biophys Biochem Cytol. 1960 Dec;8:617–638. doi: 10.1083/jcb.8.3.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. SHELDON H., ZETTERQVIST H., BRANDES D. Histochemical reactions for electron microscopy: acid phosphatase. Exp Cell Res. 1955 Dec;9(3):592–596. doi: 10.1016/0014-4827(55)90092-6. [DOI] [PubMed] [Google Scholar]
  24. SJOSTRAND F. S., HANZON V. Membrane structures of cytoplasm and mitochondria in exocrine cells of mouse pancreas as revealed by high resolution electron microscopy. Exp Cell Res. 1954 Nov;7(2):393–414. doi: 10.1016/s0014-4827(54)80086-3. [DOI] [PubMed] [Google Scholar]
  25. STAUBLI W. [New water-soluble inclusion substance for electron cytology]. C R Hebd Seances Acad Sci. 1960 Feb 8;250:1137–1139. [PubMed] [Google Scholar]
  26. SWIFT H. Studies on nuclear fine structure. Brookhaven Symp Biol. 1959 Nov;12:134–152. [PubMed] [Google Scholar]
  27. WACHSTEIN M., MEISEL E., FALCON C. Histochemistry of thiolacetic acid esterase: a comparison with nonspecific esterase with special regard to the effect of fixatives and inhibitors on intracellular localization. J Histochem Cytochem. 1961 May;9:325–339. doi: 10.1177/9.3.325. [DOI] [PubMed] [Google Scholar]
  28. WACHSTEIN M., MEISEL E. Histochemistry of hepatic phosphatases of a physiologic pH; with special reference to the demonstration of bile canaliculi. Am J Clin Pathol. 1957 Jan;27(1):13–23. doi: 10.1093/ajcp/27.1.13. [DOI] [PubMed] [Google Scholar]
  29. WATSON M. L., ALDRIDGE W. G. Methods for the use of indium as an electron stain for nucleic acids. J Biophys Biochem Cytol. 1961 Nov;11:257–272. doi: 10.1083/jcb.11.2.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. WATSON M. L. Staining of tissue sections for electron microscopy with heavy metals. II. Application of solutions containing lead and barium. J Biophys Biochem Cytol. 1958 Nov 25;4(6):727–730. doi: 10.1083/jcb.4.6.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. WATSON M. L. Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol. 1958 Jul 25;4(4):475–478. doi: 10.1083/jcb.4.4.475. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES