Publication: Double-sided staining with a gold probe and silver enhancement to detect a-amylase and sugar moieties in the mouse salivary glands
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Date
1999
Authors
Menghi, Giovanna ; Marchetti, L. ; Bondi, A.M. ; Accili, Daniela ; Sabbieti, M.G. ; Materazzi, G.
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Publisher
Murcia : F. Hernández
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DOI
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info:eu-repo/semantics/article
Description
Abstract
In the present study we report the
development of an ultrastructural electron microscopic
double-sided staining technique that, using gold probes
of 10 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 was incubated with an antibody directed to a -
amylase, followed by a secondary gold-labeled antibody,
amplification of gold particles, drying and carbon
coating; subsequently, the reverse face of the same grid,
was processed for lectin cytochemistry, with and without
sialidase digestion, and it was incubated with HRPconjugated
lectins, anti-HRP antibody and protein-A
gold. Also the reverse sequence of steps and
amplification of gold signal after the first or second
labeling were experimented. The resultant small and
large particles revealed different distributional patterns
of antigenic sites on the opposite faces of the same tissue
section. The transparency of the resin-embedded
ultrathin sections in the electron beam allowed the
simultaneous visualization of the gold probes of different
sizes present on the two faces. The analysis of
immunolabeling revealed that the a-amylase is chiefly
secreted by the parotid and submandibular glands. The application of this double-sided staining technique also
indicated that, when present in glycosylated form, the aamylase
enzyme does not contain sialic acid in the
submandibular and sublingual glands; conversely, its
location on the electron-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 0-galactose, was
found to be located.
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