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Double-sided staining with a gold probe and silver enhancement
to detect a-amylase and sugar moieties in the mouse salivary glands
G. Menghi, L. Marchetti, A.M. Bondi, D. Accili,
M.G. Sabbieti and G. Materazzi
Department of Comparative Morphological and
Biochemical Sciences, University of Camerino, Camerino, Italy
Offprint requests to: Prof.
Giovanna Menghi, Dipartimento di Scienze Morfologiche e Biochimiche
Comparate, Università di Camerino, Via Gentile III da Varano,
I-62032, Camerino (MC), Italy. Fax: (737) 402708
Summary. 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 HRP-conjugated 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 a-amylase 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 ß-galactose, was found
to be located. Histol. Histopathol. 14, 687-695 (1999)
Key words: a-amylase,
carbohydrates, lectins, immunogold, silver enhancement, salivary
glands, mouse
DOI: 10.14670/HH-14.687
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