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Advanced Histological Techniques

Fixing Tissue for Electron Microscopy

The most popular fixatives for TEM work are aldehydes and osmium tetroxide. Aldehyde based fixatives react with amines and other nucleophiles in the tissue, most notably lysine and arginine, generating cross-linked proteins. The cross linking action of these fixatives stabilizes the cytosol, preserving cellular structures.

Aldehydes do not react with most lipids, so membrane components may be leached during fixing and processing. Glutaraldehyde has been used extensively. It is a protein cross linker, penetrates small tissue blocks acceptably (2-3mm penetration in 10 hours) and gives excellent fine structure preservation. However, its main drawback is that it is a potent allergen, hazardous at levels above 0.05mg/m3 (this is a mandated limit in some EU countries).

Formaldehyde has been used for some purposes, but preservation of fine structure is not as good. One major benefit of formaldehyde fixatives is that formaldehyde is not as strongly denaturing to proteins as glutaraldehyde. Formaldehyde is therefore popular in immunohistochemistry.

Mirsky’s Fixative from National Diagnostics is similar in action to glutaraldehyde, but Mirsky’s Fixative is not allergenic and poses a reduced health hazard. Mirsky’s Fixative is a good cross-linker and gives excellent fine-structure preservation. Its speed of action can be greatly increased by the use of microwave enhancement.

Osmium tetroxide fixes tissue by cross linking lipids. It reacts with unsaturated lipids and possibly with some proteins. However most proteins are not well fixed by Osmium and will leach out of the sample during processing. Osmium tetroxide may be used as a post- or secondary fixative after an aldehyde is used. This will stabilize the membrane structures and also adds electron dense material to the section, enhancing contrast.

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