Publication: Osteopontin is histochemically detected by the AgNOR acid-silver staining
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Date
2008
Authors
Gaudin-Audrain, Christine ; Gallois, Yves ; Pascaretti-Grizon, Florence ; Hubert, Laurent ; Massin, Philippe ; Baslé, Michael-Félix ; Chappard, Daniel
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Publisher
Murcia : F. Hernández
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DOI
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info:eu-repo/semantics/article
Description
Abstract
Silver nitrate staining of decalcified bone
sections is known to reveal osteocyte canaliculi and
cement lines. Nucleolar Organising Regions (NOR) are
part of the nucleolus, containing argyrophilic proteins
(nucleoclin/C23, nucleophosmin/B23) that can be
identified by silver staining at low pH. The aim of this
study was to clarify the mechanism explaining why
AgNOR staining also reveals osteocyte canaliculi.
Human bone and kidney sections were processed for
silver staining at light and electron microscopy with a
modified method used to identify AgNOR. Sections
were processed in parallel for immunohistochemistry
with an antibody direct against osteopontin. Protein
extraction was done in the renal cortex and decalcified
bone and the proteins were separated by western
blotting. Purified hOPN was also used as a control.
Proteins were electro-transferred on polyvinylidene
difluoride membranes and stained for AgNOR proteins.
In bone, Ag staining identified AgNOR in cell nuclei, as
well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver
deposits were also observed in cytoplasmic granules on
the apical side of the cells. Immunolocalization of
osteopontin closely matched with all these locations in
bone and kidney. Ag staining of membranes at low pH
revealed bands for NOR proteins and 56 KDa (kidney),
60KDa (purified hOPN) and 75 KDa (bone) bands that
corresponded to osteopontin. NOR proteins and
osteopontin are proteins containing aspartic acid rich
regions that can bind Ag. Staining protocols using silver
nitrate at low pH can identify these proteins on
histological sections or membranes.
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