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Histology FAQ
Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
London, Canada
FAQ Home > Fixation, freezing etc
Perfusion fixative for electron microscopy
Question.
What is a suitable fixative for vascular perfusion of rats, and subsequent electron microscopy of tissues?
Answer.
A neutral, buffered, isotonic formaldehyde-glutaraldehyde mixture should be fine for any kind of electron microscopy. Many workers like to use paraformaldehyde as the source of formaldehyde.
A classical mixture is M. J. Karnovsky's (J. Cell Biol. 27: 137A-138A, 1965). This is probably the most-cited unrefereed abstract! It contains approximately 4% formaldehyde and 5% glutaraldehyde in approximately 0.1 M phosphate or cacodylate buffer. Final pH = 7.2. If cacodylate (toxic!) is used, add calcium chloride (0.5 mg/ml) to improve preservation of membrane phospholipids.
Probably this fixative is frequently misquoted, and the literature is full of references to "half-strength Karnovsky," which probably means half the glutaraldehyde concentration. A glutaraldehyde concentration of 1 to 2% is commonly considered adequate in mixtures of this kind.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
(kiernan[AT]uwo.ca)