Fixation

The choice of fixative should be determined by the chemical affect of the fixing agent on the cell component(s) to be studied.

1. Proteins: formaldehyde, in the form of neutral buffered formalin, paraformaldehyde or more recently formal alcohol or zinc formalin, are most commonly used for light microscopy. Another aldehyde, glutaraldehyde is unsurpassed in its ability to cross-link and preserve tissue proteins for routine morphological electron microscopy. Osmium also fixes most proteins but can degrade structure with prolonged exposure (+2 hrs) and decreases biological activity.

2. Nucleic Acids: picrates or mercurials are good for light microscopy. Nucleic acids are thought to react little with either glutaraldehyde or osmium directly, and are believed to be revealed by the interaction of the fixative with proteins or lipids associated with nucleic acids or by staining with uranyl salts.

3. Phospholipids: reaction with aldehydes is minimal if at all. However, they do react strongly with osmium tetroxide.

4. Polysaccharides: do not react with either osmium or glutaraldehyde, but are insoluble, so may be preserved in sections as light areas. Some types of polysaccharides can be stained prior to or during fixation with ruthenium compounds or alcian blue.

5. Glycogen: insoluble but also reacts with aldehydes.

6. Unsaturated fats: react vigorously with osmium

7. Saturated fats: do not react with aldehydes or osmium and are largely removed by dehydrants and embedments.

Aldehydes

Aldehydes react with many organic components but do not increase contrast or density. Advantages are they penetrate tissue rapidly, are convenient for perfusion and short-term storage of samples, useful for cytochemistry, and there are a diverse number of aldehydes.

1. Formaldehyde was initially thought not to be useful in electron microscopy due to commercial solutions containing large amounts of ethanol (15%), as well as the need for several days to ensure complete fixation. Formaldehyde solutions are now prepared in the lab just before use.

Formaldehyde is widely used in 1-10% concentrations for light microscopy. It is also useful in cytochemistry, when fast penetration into tissue is necessary, as well as a perfusion fixative in conjunction with glutaraldehyde and where glutaraldehyde may be too reactive. Primary fixation must be followed by osmication for electron microscopy. Specimens have a "similar" appearance to glutaraldehyde fixed tissues.

2. Glutaraldehyde has two reactive aldehyde groups, so it more tightly cross-links than formaldehyde and is not easily reversible. It forms methylene bridges between and within polypetide free amino groups. It is commonly used between 1-6% in buffer. However, a concentration of 0.3% for minimizing loss of activity and up to 12% for hard to fix samples have been reported. Fixation time is usually between 30-60 minutes depending on size of sample.

Disadvantages include the problem that stock solutions are not very stable as they will oxidize to glutaric acid, acrolein, ethanol and will polymerize. It must be stored in the dark and cold. For cytochemistry and immunocytochemistry or perfusion, you need to purify stock solutions by distillation or purchase it purified. Glutaraldehyde also results in low density so you have to follow with osmium. For cytochemistry you may have to shorten fixation times to accommodate particular reaction techniques.

3. Acrolein (acrylic aldehyde) is the most rapidly penetrating of the aldehydes. It is used for larger pieces of tissue, plant cells and microorganisms with dense walls when formaldehyde or glutaraldehydes are not suitable. Disadvantages include that it is extremely toxic, it solubilizes lipids, destroys enzyme activity, may degrade cell structures (ex- microtubules), and it has a low density which you have to follow with osmium.

4. Osmium Tetroxide (molecular weight 254.2) reacts strongly with phospholipids, unsaturated fats and sulfhydral groups of proteins. Limitations are that it is a poor penetrant (0.5mm/hr), so very small tissue pieces are needed. It also reacts with metals, has toxic vapors, is difficult to use for perfusion and it destroys enzyme activity. Currently osmium is used as a post-fixative in electron microscopy to increase the coverage of fixation, increase electron density, and to improve mechanical stability for sectioning.

5. Coagulant fixatives are also used in light microscopy. Examples include picric acid, acetic acid, mercuric chloride and methyl alcohol.

6. Other Chemical Fixatives include Glutaraldehyde/Osmium (6%/1%) which minimizes lipid extraction and cell shrinkage. (Must be used on cracked ice and freshly mixed.) Osmium/Potassium Ferrocyanide increases contrast of membranes, glycogen and microtubules. Glutaraldehyde/Acrolein enhances penetration. Glutaraldehyde/Paraformaldehyde enhances penetration. 0.2% Digitonin with 1% OsO4, 2.5% glutaraldehyde is used to retain free cholesterol. Dimethyl sulphoxide (DMSO) accelerates penetration of aldehydes. Alcian blue, lanthanum and ruthenium red react with polysaccharides. Uranyl acetate is used as a third fixative in TEM to stabilize nuclear material and phospholipids. Tannic acid/glutaraldehyde enhances contrast of the cytoskeleton.