The Cerium-Based Method is Advantageous in Combination of Phosphatase Enzyme Cytochemistry with Post-Embedding Immunocytochemistry.

Abstract

In order to expand the possibility combining enzyme cytochemistry and immunocytochemistry, the post-embedding immunogold technique was applied to tissue sections stained by either the cerium-based or lead-based method for phosphatases. After enzyme cytochemical staining for alkaline phosphatase (ALPase) and acid phosphatase (ACPase), rat liver sections were dehydrated and embedded in the LR White without osmication. The ultrathin sections were labeled by post-embedding immunogold technique for anti-cathepsin D.Both the lead-based and cerium-based methods were sufficient to localize enzyme activities in the tissue before the immunoreaction. However, lead phosphate deposits, which were reaction products of phosphatases detected by the lead-based method, largely disappeared from the tissue embedded in LR White during the post-embedding immunoreaction. In contrast, cerium phosphate deposits, reaction products by the cerium-based method, were retained in the tissue even after the immunoreaction. Moreover, from the clear-cut difference in particle sizes, the cerium phosphate precipitates were readily distinguished from the immunogold particles within the same organelle. The preceding enzyme cytochemical procedures by the cerium-based method did not hinder the sensitivity and specificity of post-embedding immunogold labeling. These results indicate that the cerium-based method has the advantage of combining pre-embedding enzyme cytochemistry with post-embedding immunocytochemistry.