A Simplified Method of Staining Thin Sections of Biological Material with Lead Hydroxide for Electron Microscopy

A method of staining thin sections with lead hydroxide which enhances both resolution and contrast was described by M. L. Watson (1958) and is now undoubtedly in routine use in many laboratories. It is the purpose of this report to present a simplification of this method which eliminates the lengthy procedure of preparing the lead hydroxide reagent and the necessity of maintaining a COrfree atmosphere during the staining interval. On exposure to air, lead hydroxide forms lead carbonate, which may contaminate the specimen grid. In the procedure presented here it is believed that the reaction leading from lead acetate to lead hydroxide occurs in the specimen section. In any case it was only on rare occasions that a minimal amount of fine precipitate, presumably of lead carbonate, could be observed in the tissue. The tissue represented in the accompanying plates was prepared in the following manner. The pancreas of an 8 day old posthatched chick was diced and fixed for 1 hour in chrom-osmium solution (Dalton, 1955; Zeigel, in press). The small blocks of tissue were then immersed in 10 per cent formalin for 1 hour and dehydrated in a graded series of ethanols. They were infiltrated with, and embedded in, a 3:1 mixture of n-butyl to ethyl methacrylate which was catalyzed by 0.25 per cent benzoyl peroxide at 80°C. Sections were cut with a Porter-Blum microtome using a diamond knife and picked up from a 30 per cent acetone-in-water solution on formvar-coated Athene type grids. The sections are stained in the following manner. A saturated solution of lead acetate in boiled distilled water is maintained in a glass stoppered bottle so that a visible number of undissolved crystals remain at the bottom of the bottle. After several drops of the solution have been removed, the reagent bottle should be refilled to the top with boiled distilled water, thus eliminating any air pocket at the top of the bottle. I t is thought that this prevents crystals of lead carbonate from forming on the surface of the reagent and thus decreases the possibility of contaminating the grids. With a