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.