FIXATIVES

(Taken from Dr. Steffen’s Notes)

Formaldehyde

* A colorless, irritating gas most familiar as commercial grade formalin, a 37-40% aqueous solution of
formaldehyde and up to 15% methanol added to inhibit polymerization into paraformaldehyde.
* Value as a tissue preservative known for over a century, thus its common inclusion in many of the old pickling recipes.
* Mode of action - forms bonds with the amino, imino, and peptide groups of proteins which in turn react with active hydrogens atoms of other groups, forming cross-links between peptide chains (thus it is referred to as an additive fixative. Rxn w/ proteins is affected by factors such as concentration, pH, temperature, and duration fixation. An increase in any of these factors results in an increase in binding by formaldehyde.
* Many of the reactions with proteins are reversible.
* Rxn w/ other compounds - very effective in stabilizing nucleic acids and nucleoproteins (thus its wide use in fixation of bacteria and viruses. Less effective in stabilizing carbohydrates, no effect on soluble carbohydrates, but does stabilize glycogen and mucoproteins. Reacts w/ lipids but does not stabilize them.
* Formalin contains many impurities, so formaldehyde for use in EM is normally prepared from the dissolution, heating, and alkalination of powdered paraformaldehyde. Since this solution contains no inhibitors, it has a shelf life of only a few weeks.
* Formaldehyde is the least cross-linking and thus the least stabilizing of all the aldehydes, thus is usually used in conjunction with other aldehydes such as GTA It is useful in procedures where minimal protein conformational change is desired, such as in immunolocalization and enzyme localization studies.
* Low MW makes it one of the best penetrating of all the fixatives, thus it is widely used in fixation of resistent materials, such as seeds, spores, plant material, etc., usually in conjunction w/ another aldehyde.

Glutaraldehyde (GTA)

* Glutaric acid dialdehyde, a 5 Carbon dialdehyde, is the most widely applied fixative in both scanning and transmission electron microscopy. Tn TEM, buffered GTA has the reputation of providing the best ultrastructural preservation in the widest variety of tissue types of any known chemical fixative.
* Most highly cross-linking of all the aldehydes.
* Mode of action - Rxn w/ proteins is not fully understood. Known to involve lysine side chains of proteins, and that GTA fixation is irreversible.
* Rxn w/ other components - not studied to any great extent. Does not prevent lipid extraction. Nucleic acids not preserved directly, but may be retained due to the preservation of associated proteins in eukaryotes. Bacterial and viral DNA not preserved. Soluble carbohydrates not preserved, but some glycogen may be retained.
* Commercial grade GTA is usually a 25 or 50% aqueous solution with a pH between 3 and 6, and containing various impurities such as ethanol, methanol, glutaric acid, and various polymers and exidation products. These products can exert a considerable influence on fixation. Some lots of GTA are known to contain higher quantities of impurities than aldehyde. For this reason, it is advisable to use high quality distilled GTA prepared specifically for EM, and sealed in glass ampules under inert gas, particularly in applications where consistency and a high degree of control in fixation is desired.

Acrolein (acrylic aldehyde)

* A 3 carbon olefinic aldehyde, introduced to EM by Luft (1959) for its utility as a rapidly penetrating primary fixative in applications where penetration tends to be a problem. It is a highly volatile and reactive compound, features that may be desirable in certain applications, and at the same time give it a reputation for being hazardous and difficult to handle. Smells like scorching fat.
* Volatility gives it application as a vapor fixative, especially in cases where anhydrous fixation is necessary, such as in the stailization of mineralized tissues where mineral loss or translocation may be a problem.
* Mode of action - reacts rapidly with proteins, forming crosslinks at amino, imino, sulfhydryl, and imidazole groups. Also has ability to stabilize lipids, probably as a result of forming crosslinks with adjacent proteins.
* Commercial grade acrolein is available as a 100% colorless flammable liquid. It has an unpleasant acrid odor, with vapors extremely irritating to mucosal surfaces at concentrations as low as 1 ppm. It is a component of tear gas. Fume hoods, gloves, and protective clothing are advisable when using it. Better still, use something else.

Osmium Tetroxide

* Osmium tetroxide (OsO4, osmic acid) has remained an almost universal standard in preparative procedures for TEM since its introduction for that purpose over 3 decades ago. Its principle utility in this respect is its ability to stabilize lipids, as a stain, and as a mordant for other stains. Although it is widely used in preparative schemes for SEM, this must be due at least in part to arbitrary whole-cloth adoption of TEM fixation schemes for SEM. Except for cases where lipid retention is essential, the aforementioned qualities of this compound have much less to offer the area of SEM.
* A non-polar tetrahedral molecule with a molecular weight of 254 and solubility water and a variety of organic compounds. Commercially available as a coarse yellow crystalline material packaged in glass ampules sealed under inert gas. Similarly packaged aqueous solutions are also available.
* An additive, non-coagulative type of fixative, but lacks the ability to crosslink many proteins. Given this, and fact that it has a very poor rate of penetration, its use as a primary fixative is quite limited, although it is popular in some mixtures with other fixatives for unicellular organisms. Due to its extreme toxicity, low vapor pressure, and being a strong oxidizing agent, precautions are necessary for its handling.
* Mode of action - reacts primarily w/ double bonds and sulfhydryl groups of proteins, causing major conformational changes in the 1E and 2E structure of proteins...unsuitable for studies involving immunolocalization or enzyme activity.
* Properties Making it Useful in SEM - Causes tissue hardening, imparting mechanical strength and affording some extra resistance to shrinkage and distortion during subsequent steps. Imparts a small measure of conductivity, which is useful in specimens with extremely detailed surfaces or with deep recesses such as the crypts between intestinal villi, which are not easily coated. Also serves as a mordant for some of the chemical coating deposition techniques which will be mentioned later.

PICRIC ACID

* Trinitrophenol - a key ingredient of Bouins solution, a classic coagulative fixative. Picric acid is not itself coagulative, but precipitates proteins, rendering them insoluble without altering their structure. Many labs routinely employ it with aldehydes as a general purpose fixative.
* Results in considerable higher retention of proteins
* Related to TNT (trinitrotoluene), and is a powerful explosive. It may be safely kept in the lab if kept wet. A saturated aqueous solution is maintained for addition to buffered aldehydes to a final concentration of 0.25 - 1%.