Double-sided staining with a gold probe and sliver enhancement to detect alpha-amylase and sugar moieties in the mouse salivary glands

In the present study we report the development of an ultrastructural electr on microscopic double-sided staining technique that, using gold probes of 1 0 nm and enhancement of the gold signal by silver amplification, allows the demonstration of two antigenic sites on the same section. The labeling was carried out in the following manner: one face of uncoated floating grids w as incubated with an antibody directed to alpha-amylase, followed by a seco ndary gold-labeled antibody, amplification of gold particles, drying and ca rbon coating; subsequently, the reverse face of the same grid, was processe d for lectin cytochemistry, with and without sialidase digestion, and it wa s incubated with HRP-conjugated lectins, anti-HRP antibody and protein-A go ld. Also the reverse sequence of steps and amplification of gold signal aft er the first or second labeling were experimented. The resultant small and large particles revealed different distributional patterns of antigenic sit es on the opposite faces of the same tissue section. The transparency of th e resin-embedded ultrathin sections in the electron beam allowed the simult aneous visualization of the gold probes of different sizes present on the t wo faces. The analysis of immunolabeling revealed that the cr-amylase is ch iefly secreted by the parotid and submandibular glands. The application of this double-sided staining technique also indicated that, when present in g lycosylated form, the alpha-amylase enzyme does not contain sialic acid in the submandibular and sublingual glands; conversely, its location on the el ectron-dense areas of target granules in the parotid acinar cells seems to suggest that a sialylated isoenzymatic form can occur within these granule regions where sialic, acid linked to beta-galactose, was found to be locate d.