Localization of methylene blue paramolybdate in vitally stained nerves

Methylene blue taken up by living neurons can be preserved for electron microscopy in a fixative containing osmium tetroxide and ammonium paramolybdate at pH 5.2. Paramolybdate is the buffer, precipitating agent and main osmotic ingredient; it does not function as an electron stain unless methylene blue is present. The low pH keeps the dye/paramolybdate complex from dissolving. Neither the low pH nor drastic dehydration from water to absolute ethanol harm the tissue. The staining mechanism involves cationic methylene blue associating with anionic structures such as microtubules and neurofilaments in the living cell; during fixation paramolybdate forms a precipitate with the dye at the staining sites. This fixative does not preserve microtubules unless they are first vitally stained.