Immunohistochemical detection of active transforming growth factor-β in situ using engineered tissue

Abstract

The biological activity of transforming growth factor-β1 (TGF-β) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-β could provide insight into TGF-β activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-β status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-β or constitutively active TGF-β. This comparison revealed that, whereas most antibodies did not differentiate between TGF-β activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-β, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-β versus latent TGF-β in cryosections. However, in formalin-fixed, paraffin- embedded tissue, discrimination of active TGF-β by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-β. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-β only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-β in both frozen and paraffin-embedded tissues. Thus, in situ discrimination of active versus latent TGF-β depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.