IMPROVED IMMUNOHISTOCHEMICAL STAINING OF OSTEOPONTIN (OPN) IN PARAFFIN-EMBEDDED ARCHIVAL BONE SPECIMENS FOLLOWING ANTIGEN RETRIEVAL - ANTI-HUMAN OPN ANTIBODY RECOGNIZES MULTIPLE MOLECULAR-FORMS

Citation
Re. Devoll et al., IMPROVED IMMUNOHISTOCHEMICAL STAINING OF OSTEOPONTIN (OPN) IN PARAFFIN-EMBEDDED ARCHIVAL BONE SPECIMENS FOLLOWING ANTIGEN RETRIEVAL - ANTI-HUMAN OPN ANTIBODY RECOGNIZES MULTIPLE MOLECULAR-FORMS, Calcified tissue international, 60(4), 1997, pp. 380-386
Citations number
25
Categorie Soggetti
Endocrynology & Metabolism
ISSN journal
0171967X
Volume
60
Issue
4
Year of publication
1997
Pages
380 - 386
Database
ISI
SICI code
0171-967X(1997)60:4<380:IISOO(>2.0.ZU;2-Z
Abstract
Studies to assess osteopontin (OPN) localization in adult human bone u sing immunochemical techniques produce conflicting results due to vari ations in tissue processing or antibody immunoreactivity. The present study was designed to resolve these discrepancies using well-character ized antibodies and improved antigen detection. An anti-osteopontin (a lpha-OPN) antiserum was developed that recognizes various soluble mole cular weight forms of human OPN, including monomeric, cleaved, and dim erized products. An affinity column of full length recombinant human O PN (rOPN) coupled to support was used to purify alpha-OPN antibodies. Western analysis showed that the affinity-purified antibodies recogniz ed numerous molecular weight forms of OPN. These antibodies were used to study the distribution of OPN in adult human bone using immunohisto chemical techniques combined with an antigen retrieval protocol utiliz ing a newly developed antigen retrieval solution, Retriev-All(TM) (Bro nco Technologies Inc, Pasadena, TX). Immunolocalization of OPN in arch ival bone specimens prior to antigen retrieval produced no demonstrabl e immunostaining even at high concentrations of alpha-OPN. Use of the antigen retrieval protocol restored OPN immunoreactivity, with strong staining apparent in cement lines, osteoblasts, osteocytes, canaliculi , osteoid, and bone matrix. We conclude that antigen retrieval restore s immunochemical recognition of OPN in archival specimens containing b one without increasing nonspecific binding.