Journal of Histotechnology ISSN: 0147-8885 (Print) 2046-0236 (Online) Journal homepage: http://www.tandfonline.com/loi/yhis20 Introduction to the Theory and Practice of Fixation of Tissues Isam Eltoum, Jerry Fredenburgh, Russell B. Myers & William E. Grizzle To cite this article: Isam Eltoum, Jerry Fredenburgh, Russell B. Myers & William E. Grizzle (2001) Introduction to the Theory and Practice of Fixation of Tissues, Journal of Histotechnology, 24:3, 173-190 To link to this article: http://dx.doi.org/10.1179/his.2001.24.3.173 Published online: 18 Jul 2013. Submit your article to this journal Article views: 93 View related articles Citing articles: 35 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yhis20 Download by: [the Bodleian Libraries of the University of Oxford] Date: 01 April 2016, At: 03:05 Introduction to the Theory and Practice of Fixation of Tissues - Isam ~ltoum',Jerry ~redenbur~h',Russell B. ~~ers"William E. ~rizzle"" ' Department of Pathology, University of Alabama at Birmingham, Birmingham, AL ' Richard Allan Scientific, Kalamazoo, MI Abstract croscopic relationships among cells, cellular components Many approaches to fixation and types of fixatives have (eg, cytoplasm, nuclei), and extracellular material, with been develo~edand tested over the last-centurv. The mecha- little disruption of the organization of the tissue and with nisms by which fixatives act to harden and preserve tissues maintenance of the tissue's local chemical composition. Be- fall into broad categories, including dehydrants, heat ef- cause many components of tissues are soluble in aqueous or fects, cross-linkers, effects of acids, and combinations of other liquid environments, a reliable picture of the micro- these categories. Each fixative has advantages and disad- anatomy and microenvironment of the tissue requires that vantages, including specific lnolecules retained within sol~iblecomponents not be lost fro111 stained tissue sections. "fixed" tissues, swelling or shrinkage of fixed tissues, varia- For example, if sol~~blecornponents are lost from the cyto- tions in the a~lalitvof histoche~nicaland in~munohistochem- A d plasm of cells, the color of the cytoplas~non he~natoxylin ical staining, and varying capabilities to nlaintain the struc- and eosin (H&E) staining will be reduced or nlodified and tures of cellular organelles. One of the major problems with aspects of the appearance of the microanatomy of the tissue formaldehyde type (cross-linking) fixatives has been the will be lost or changed. loss of antigen immunorecognition; correcting this usually Allnost any method of fixation induces shrinkage, swell- requires some method of antigen recovery. Similarly, the ing, and hardening of tissues, and color variations with vari- extraction of lnRNA and DNA from formalin fixed tissue in ous histochemical stains (1-5). To maintain the microarchi- paraffin blocks is problematic. All widely used fixatives are tecture of tissue along with as much of the soluble selected by compron~ise-good aspects are balanced against components of tissue as practicable, a "fixative" is used to less desirable features. This article disc~issesthe basics of mini~nizethe loss of cellular components, including pep- fixation and provides the formulas for the fixatives currently tides, proteins, lipids, mRNA, and DNA and to prevent the used in pathology, histology, and anatomy and discusses destruction of ~nacron~olecularstructures such as cytoplas- good and bad aspects of specific fixatives. (Tlze J Histotech- mic membranes, ro~lghendoplasmic reticulum, smooth en- rzol 24: 173, 200 1) doplasmic reticulum, nuclear membranes, mitochondria, and lysosomes. By minimizing the loss or enzytnatic de- Key words: antigen recovery, cross-linkage, dehydration, struction of cellular and extracellular ~nolec~ilesand by formaldehyde, heat artifacts maintaining lnacromolecular structures and protecting tis- sues from destruction by microorganisms, the fixative acts Introduction to "fix" cit a poirzt bz ritne one view of a dynamically chang- From embalming to electron microscopy, the major ob- ing, viable tissue. A fixative should also prevent the subse- jective of fixation has been to maintain excellent morpho- quent breakdown of the tissue by enzymatic activity andlor Downloaded by [the Bodleian Libraries of the University Oxford] at 03:05 01 April 2016 logical features. The use and develop~nentof specific fixa- by lnicroorganisrns during long-term storage. tives usually have been empirical, and in biological sciences A fixative not only interacts initially with the tissue in the have borrowed fixation information and techniques from tissue's aqueous environment, but subsequently the fixative such industries as leather tanning and vaccine production. In and the chemical modifications induced by the fixative have order to understand visually the microanatolny of tissues, additional activity and change the ~nolecularfeatures of the stained microscopic tissue sections must maintain the mi- tissue during all phases of tissue processing and staining, from dehydration of the tissue to staining of tissue sections using histoche~nicalor irnn~unohistochemicalstains. Fixa- Address and reprint requests, Dr. William E. Grizzle, Department of tion, tissue processing, and staining protocols used in the Pathology, University of Alabama at Birmingham, Zeigler Research Build- preparation of stained slides produce a cornpro~nisedde- ing, Room 422, 703 So~ith19th Street, Birmingham, AL 35233-0007. Phone: (205)-934-42 14; FAX: (205)-975-7 128; E-mail: grizzle@path.~~ab. piction of features that represent the original living tissue. edu Each fixative and tissue processing protocol maintains some The Journal of Hlstotechnology I Vol. 24, No. 3 I September 2001 molecular and ~nacro~nolecularaspects of the tissue better The best examples of such cross-linking fixatives are form- than some other fixative-processing combinations. Presently, a aldehyde and glutaraldehyde. Another biochemical ap- universal or ideal fixative has not been identified; fixatives are proach to fixation is the use of agents that remove free water selected currently on the basis of producing a final product from tissues and hence precipitate and coagulate proteins. needed to demonstrate a specific feature of a tissue. Examples of such fixatives include dehydrants such as etha- The most important aspect of a fixative is its ability to nol, methanol, and acetone. Other fixatives may rely on prevent short- and long-term destruction of the microarchi- denaturing proteins and nucleic acids through changes in pH tecture of the tissue. Fixation prevents the short- and long- or via salt formation. Examples of such fixatives are acetic term destruction of the microarchitecture by stopping the acid, trichloroacetic acid, and zinc acetate. Some complex activity of catabolic enzymes (stop autolysis), stopping the fixatives are mixtures of several types of fixatives. For ex- activity of microorganisms (stop tissue degradation, and ample, alcoholic formalin acts to fix tissues by cross-linking minimizing the diffusion of soluble substances from their as well as by dehydration. original locations. Other important characteristics of a good fixative include the destr~iction of infectious agents, the Physical Methods of Fixatioiz maintenance of tissue and cellular integrity, and possession Heat Fixatiorz ,- of a good toxicological and flammability profile that per- Probably, the simplest form of fixation is "heat." When mits the safe use of the chemical (6). The advent of new we boil or poach an egg, we use heat to precipitate proteins biological methods, the increased understanding of the hu- within the egg, so when cutting the egg we can identify the man genome, and the need to evaluate rapidly the biology of yolk and egg white separately. Each of these components is disease processes have placed new demands on the old fixa- less soluble in water after heat fixation than they are as tion processes. Fixatives used in academic environments components of a fresh egg. Picking up a frozen section on should permit the recovery of macromolec~~les,including a warm microscope slide attaches the section to the slide, proteins, mRNA, and DNA without extensive biochemical partially fixes (by heat) the tissue section, and partially de- modifications. Another important characteristic of an ideal hydrates the tissue section. Although reasonable morphol- fixative is the versatility to be used with a wide variety of ogy could be obtained by boiling tissue in normal saline, in tissue from humans as well as many other different species histopathology, heat fixation alone is not used frequently; of animals. It is equally important that the ideal fixative rather heat is used to accelerate other forms of fixation as penetrate both small and large specimens rapidly and pre- well as tissue processing. serve fixed tissue in paraffin blocks for at least a decade. Microwave Fixation Fixative compatibility with all automatic tissue processors Microwave heating is used to speed fixation reducing the is a must as well as the ability to support excellent mi- time required for fixation of gross specimens and histologi- crotomy of paraffin blocks (7). The ideal fixative should cal sections from more than 12 hr to less than 20 min for promote excellent staining with H&E and also allow histo- some specimens (9,lO).Microwaving tissue in formalin re- chemical, im~nunohistoche~nical,and