A new fixative for the preservation of actin filaments: fixation of pure actin filament pellets.

Abstract

Conventional fixation for thin-section microscopy is insufficient to preserve many elements of cells and tissues. Actin filaments, for example, are destroyed during post-fixation in OsO4. In our search for a better fixative, we chose pellets of pure actin filaments as a very sensitive model system. In the present study, the potential of amines for improving aldehyde fixation was explored, and the results were compared to those obtained with the use of tannic acid. Aldehyde and amine were used together as an initial fixative, followed by aldehyde alone with postfixation in 1% OsO4 in buffer at 4 degrees C for 15 min, uranyl acetate en bloc stain, acetone dehydration, and embedding in Epox 812. Some primary monoamines improved the preservation of filaments; filaments were not broken beyond recognition by OsO4, as occurs when glutaraldehyde alone is used. Excellent preservation was seen when certain primary diamines were used. The quality of this fixation was superior to that obtained with tannic acid and was without the large increase in filament diameter that is seen with concentrations of tannic acid sufficient to protect filaments against osmium damage. The effects on filaments of the amines lysine, putrescine, ammonium, and arginine have been documented in detail, as we systematically varied all the major parameters normally considered in formulating fixation protocols.