Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
What is the best fixative for frozen sections?
Unfixed tissue, cut with a cryostat (thin sections) or a vibrating microtome (thick sections) should be fixed if this is compatible with the staining technique to be used.
Many enzyme histochemical methods demand unfixed sections, and so do immunohistochemical methods with some (fortunately not most) primary antibodies. Enzyme incubations are often terminated by moving the slide or coverslip bearing the cryostat section from the incubation medium into a neutral, buffered formaldehyde fixative.
Even "minimal" (= inadequate) fixation before staining will greatly improve the structural preservation of tissue. Many enzymes will survive either a minute or two in neutral, buffered formaldehyde, followed by a wash in buffered saline. Some enzymes and most antigens will survive immersion of the slide or coverslip in cold (about 0 C) acetone for half a minute. The acetone is allowed to evaporate before immersing the section in incubation medium.
Cryostat sections may also be fixed by heating, but this inactivates most enzymes. A drop of an ethanol-poly(ethylene glycol) mixture is placed on the section and the temperature brought up to 55 C in a microwave oven. (A special laboratory oven is needed to get this amount of control.)
Kiernan JA 1999. Histological and histochemical Methods, 3rd ed. Oxford: Butterworth-Heinemann.
Kok LP & Boon ME 1992. Microwave Cookbook for Microscopists. Leiden: Coulomb Press.
Pearse AGE 1980. Histochemistry, 4th ed. Vol 1.
John A. Kiernan
Department of Anatomy,
The University of Western Ontario,
LONDON, Canada N6A 5C1