A new cytochemical method: Combined enzyme cytochemistry and immunocytochemistry on ultrathin cryosections

Cryo-ultramicrotomy has been a useful method for immunocytochemistry because it can preserve the ultrastructure of cells and also display antigenic sites in cells. Recently, cryo-ultramicrotomy has been successfully applied to enzyme cytochemistry for the demonstration of some phosphatases with lead as the capture metal. However, this application has some problems. The lead reaction product is unstable with uranyl acetate staining, thus leading to poor ultrastructural detail. The purpose of this study is to develop the new method of enzyme cytochemistry using cerium as the capture metal on ultrathin cryosections labeled with immunogold probes. This method should be of importance for demonstrating the ultrastructure localization of certain enzyme and the simultaneous localization of antigens in a variety of cell types. Freshly isolated human neutrophils were fixed with paraformaldehyde and/or glutaraldehyd in 0.1 M cacodylate buffer (pH 7.2) with 5 % sucrose, then pelleted into 10 % gelatin and subsequently infiltrated with 2.3 M sucrose.