title: Improved immunohistochemical staining of osteopontin (OPN) in paraffin-embedded archival bone specimens following antigen retrieval: anti-human OPN antibody recognizes multiple molecular forms.
abstract: Studies to assess osteopontin (OPN) localization in adult human bone using immunochemical techniques produce conflicting results due to variations in tissue processing or antibody immunoreactivity. The present study was designed to resolve these discrepancies using well-characterized antibodies and improved antigen detection. An anti-osteopontin (alpha-OPN) antiserum was developed that recognizes various soluble molecular weight forms of human OPN, including monomeric, cleaved, and dimerized products. An affinity column of full length recombinant human OPN (rOPN) coupled to support was used to purify alpha-OPN antibodies. Western analysis showed that the affinity-purified antibodies recognized numerous molecular weight forms of OPN. These antibodies were used to study the distribution of OPN in adult human bone using immunohistochemical techniques combined with an antigen retrieval protocol utilizing a newly developed antigen retrieval solution, Retriev-Alltrade mark (Bronco Technologies Inc, Pasadena, TX). Immunolocalization of OPN in archival bone specimens prior to antigen retrieval produced no demonstrable 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 restores immunochemical recognition of OPN in archival specimens containing bone without increasing nonspecific binding.
authors.affiliation: Department of Basic Sciences, The University of Texas-Houston, Health Science Center, Dental Branch, 6516 John Freeman Avenue, Houston, Texas 77030, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Devoll
authors.forename: G J
authors.initials: GJ
authors.lastname: Pinero
authors.forename: E R
authors.initials: ER
authors.lastname: Appelbaum
authors.forename: E
authors.initials: E
authors.lastname: Dul
authors.forename: P
authors.initials: P
authors.lastname: Troncoso
authors.forename: W T
authors.initials: WT
authors.lastname: Butler
authors.forename: M C
authors.initials: MC
authors.lastname: Farach-Carson
mesh_terms: D000328:Adult; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D001842:Bone and Bones / Q000737:chemistry*; D005260:Female; D006041:Goats / Q000276:immunology; D006801:Humans; D007085:Ilium / Q000737:chemistry; D007106:Immune Sera; D007124:Immunoenzyme Techniques; D007684:Kidney Tubules / Q000737:chemistry; D008297:Male; D008894:Milk Proteins / Q000032:analysis; D008895:Milk, Human / Q000737:chemistry; D008970:Molecular Weight; D009928:Organ Specificity; D053495:Osteopontin; D016612:Paraffin Embedding; D011467:Prostate / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D012795:Sialoglycoproteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012996:Solutions; D013048:Specimen Handling; D016707:Tissue Fixation
chemical_list: D000906:Antibodies; D007106:Immune Sera; D008894:Milk Proteins; D011993:Recombinant Fusion Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D012996:Solutions; D053495:Osteopontin