Variations in Sample Preparation that Affect Contrast Enhancement by Lead Aspartate

Abstract

We recently described a new en bloc lead contrast stain for transmission electron microscopy (Walton, 1979). Several laboratories report great success with lead aspartate staining but some others have found that its contrast enhancement is insufficient. We undertook the present study to determine how differences in sample protocol affect lead aspartate contrast enhancement, making a strict effort to obtain comparable samples. Gastrointestinal duodenum provided a tissue of uniform diameter. A 1" length of duodenum was flushed with warm saline and fixed in 4% paraformaldehyde-1% glutaraldehyde solution buffered at pH 7.3 with 0.1 M cacodylate. After a 2 hr fixation period, the sample was cross-sectioned into blocks 1.0 mm thick. Samples were then post-fixed with 1% osmium tetroxide buffered with veronal acetate at pH 7.3, washed, stained with lead aspartate, rinsed, dehydrated, and embedded. Standard staining conditions were pH 5.5 lead aspartate heated to 60°C for 1 hr. This procedure was varied by adjusting either the pH of lead aspartate, the staining time, or the temperature. Additional variations were made in the dehydration times, osmium fixation times, and choice of wash buffer. Silver sections were cut from all samples. Micrographs were taken using uniform settings at either high or low magnifications. All negatives were developed and printed under the same conditions.