Osmium

Osmium tetroxide is one of very few compounds that actually fixes lipids. It is used for both light and electron microscopy, although more commonly for the latter because of its efficacy for lipid containing cell membranes.

In light microscopy osmium has some use for initial fixation, sometimes in conjunction with chrome compounds, another lipid fixative. It is no longer used to any degree for routine preservation, partly due to its expense, but also due to the difficulties associated with its use. It must always be used in a fume hood as it causes intense and characteristic headaches. It is possible to use osmium tetroxide as a secondary fixative, a process called post-osmication. the tissue is first fixed in a simple formalin fixative, then refixed in a simple osmium tetroxide solution. This fixes the lipids, both cell membrane bound lipid and triglycerides. After paraffin processing sections are cut and usually simply dewaxed and coverslipped, without staining. Lipids appear black, and other tissues are grey.

Method

1. Fix 2 mm thick slices of fresh tissue in 10% neutral buffered formalin for 24 hours.
2. Wash the tissue with running tap water overnight to remove all traces of formalin. Residual formalin will reduce osmium tetroxide to a black deposit during the next step.
3. Place into 0.1% aqueous osmium tetroxide for 24 hours.
4. Wash the tissue with running tap water overnight to remove all traces of osmium tetroxide. Residual osmium tetroxide will be reduced it to a black deposit during processing.
5. Place into a paraffin schedule at a convenient point. It may be placed into a formalin fixative, or ethanol.
6. After blocking out, cut 5-10µ sections, bake onto slides, dewax with xylene and coverslip using a resinous medium.

Results

• Lipids, including triglycerides  –  black
• Background  –  grey

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