A Routine Technique for Double-staining Ultrathin Sections Using Uranyl and Lead Salts

For more than 2 years, a double-staining of sections of OsO 4-fixed and Epon-embedded tissues with uranyl acetate followed by lead hydroxide has been routine technique in this laboratory. It was found to give substantially higher contrast, thus permitting the use of thinner sections. It facilitated focusing and routinely provided clearer pictures of cell fine structure than could be obtained with either stain alone. However, the formation of a fine precipitate frequently marred the sections, and this difficulty could never be satisfactorily controlled. Apparently, it is caused by the uranyl acetate solution which is not stable at the pH and concentration used. Decreasing the concentration and/or the pH of the solution caused an undesirable loss of staining intensity. Therefore, a number of different uranyl salts were tested as a substitute for uranyl acetate in the procedure, and one of them, uranyl magnesium acetate, has been found to give consistently good results.