Electron micrographs of PAF fixation show marked lipid extraction. This most likely indicates that which step was not used?

• A. Secondary osmium tetroxide fixation.
• B. Primary osmium tetroxide fixation.
• C. Bouin's fixative.
• D. Formaldehyde alone.

Answer: (A)

Lipid preservation in electron microscopy relies on osmium tetroxide as a post-fixative. Osmium binds to and stabilizes membranes by reacting with unsaturated lipids, making them electron-dense and resistant to extraction during the dehydration and embedding steps. If this osmium post-fixation step is not used, lipids are not properly fixed and are readily removed during processing, leading to marked lipid extraction in the final micrographs. Among the options, omitting the secondary osmium tetroxide fixation best explains the observed lipid loss.