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

Zinc-containing fixatives: What has been published?
(Answers include references, opinions and discussion.)


What published work is available with evaluations of zinc-formalin and other such newer fixatives? Can a zinc salt really replace mercuric chloride?


These questions are discussed quite frequently in the HistoNet listserver group. In February 1998. I wrote that there was a shortage of publications in refereed journals, and also suggested that it was unwise to use a commercial product without knowing its complete composition. (There are published formulations, but in most cases these compare a zinc-containing liquid with neutral buffered formaldehyde, for immunohistochemical detection of one or several antigens. The exact composition of proprietary fixative mixtures is rarely stated in catalogues etc.)

John Kiernan
London, Canada

Dick Dapson disagreed with some of my comments, and provided a helpful list of publications:

John Kiernan wrote (2/19/98) that there is a remarkable shortage of literature comparing zinc formalin solutions with conventional fixatives. Actually, the subject has been covered rather well over a time span of more than 10 years.  Here is a sample that shows the evolution of these remarkable fixatives; all are from refereed journals and (except for the 1981 abstract) have "passed the scrutiny of the regular scientific publication process":

1981.  Jones, et al.  Transition metal salts as adjuncts to formalin for tissue fixation (abstract).  Lab Invest 44:32A [This is the paper that really started it all, although zinc formulations do appear in the early literature].

1983.  Mugnaini et al.  Zinc-aldehyde fixation for light-microscopic immunocytochemistry of nervous tissues. J Histoch Cytochem 31:1435-1438.

1985.  Banks.  Technical aspects of specimen preparation and special studies.  In Surgical Pathology of the Lymph Nodes and Related Organs. Jaffe, ed.  W B Saunders Co., pp1-21.

1988.  Herman, et al.  Zinc formalin fixative for automated tissue processing.  J Histotechnol 11:  85-89. [The first really comprehensive study comparing NBF and unbuffered zinc sulfate formalin].

1990.  Tome, et al.   Preservation of cluster 1 small cell lung cancer antigen in zinc-formalin fixative and its application to immunohistochemical diagnosis.  Histopathol 16:  469-474.

1991.  Abbondanzo, et al.  Enhancement of immunoreactivity among lymphoid malignant neoplasms in paraffin-embedded tissues by refixation in zinc sulfate-formalin.  Arch Pathol Lab med 115:31-33.

1993.  Estrogen and progesterone receptor proteins in zinc sulfate, formalin fixed breast carcionoma:  advantages of a supersensitive streptavidin technique.  J Histotechnol 16: 51-56.

1993.  Dapson.  Fixation for the 1990's:  a review of needs and accomplishments.  Biotechnic & Histochem 68:75-82. [Like Herman's paper, this provides a critical comparison between NBF and zinc formalin; it also details probable mechanisms and reviews the pertinent literature to date].

1995.  L'Hoste, et al.  Using zinc formalin as a routine fixative in the histology laboratory.  Lab Med 26:  210-214. [Compares NBF and a buffered zinc formalin, using side-by-side color photomicrographs].

Richard W. Dapson, Ph.D.
1020 Harts Lake Road
Battle Creek, MI  49015

My response:

The interested reader should study these publications. Most do not include critical comparisons with other fixatives (except buffered formaldehyde), especially for preservation of intracellular structures. There is a real need for users to compare several fixatives in properly controlled trials, and publish their results.

Zinc mixtures became popular in the early 1990s, but the earliest (probably) of its kind was introduced soon after the fixative action of formaldehyde was discovered by F. Blum (in Germany, in 1893). This is Fish's fixative:

         Water:            2000 ml
         Formalin:           50 ml
         Zinc chloride:       15 g

    Fish, Pierre A. 1895. The use of formalin in neurology.
                          Trans. Am. Microsc. Soc. 17: 319-330.

[Fish recommended immersion of the brain for 7-10 days, with injection of cavities and blood vessels if possible. It's all been done before if you go back far enough! Fish's paper also reviewed the uses of formaldehyde (31 references, 2 years after it's introduction as a fixative) and described other fixative mixtures.]

J. A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6K 5C1