Contrast Staining Protocols for Microscopy

The most broadly useful contrast stains tested so far are inorganic iodine and phosphotungstic acid (PTA)[22 (link)]. The formulations and general procedures used are given in Table 2, and notes on the fixatives used are in Table 3[23 -25 (link)]. The stains and procedures are simple and the procedures are robust. The staining times were found not to be critical, as long as the stain had sufficient time to penetrate the tissues. Inorganic iodine in alcoholic or aqueous solution diffuses rapidly into fixed tissues and was able to stain most specimens in a few hours or less, although staining was generally done overnight. PTA is a much larger molecule [26 (link)], and the solution used here was found to require overnight incubation to penetrate specimens 2–3 mm thick, and longer for larger specimens. PTA is known to bind heavily to various proteins and connective tissue [27 ,28 ], and this property, along with electron-shell energies that match common x-ray source emissions, suggested that it might be a useful stain for x-ray imaging. A few samples were tested with phosphomolybdic acid (PMA) staining, used similarly to PTA. The results (not shown) were generally similar, and PMA was not pursued further here (but see refs. [29 (link)] and [30 ] for successful application of PMA).