THE USE OF MAB-1977 MONOCLONAL-ANTIBODY FOR THE IMMUNOHISTOCHEMICAL LOCALIZATION OF BETA-1 INTEGRINS IN PARAFFIN-EMBEDDED HUMAN KIDNEY

Aims and background: Integrins are widely known cell membrane receptor s for extracellular matrix molecules. The beta 1 integrin subgroup is mainly expressed by kidney cells; immunolocalization of these molecule s is usually carried out on cryostatic sections. A commercial monoclon al antibody directed against the human beta 1 integrin was tested in o rder to design a method for the detection of this antigen in formalin- fixed, paraffin-embedded human kidney tissue. Methods: Specimens obtai ned from nephrectomies were fixed with 10% formalin and embedded in pa raffin. Three different detection protocols were applied after incubat ion with the antihuman beta 1 integrin monoclonal antibody (MAB 1977): 1) immunoperoxidase with labelled streptavidin biotin (LSAB), using b iotinylated secondary antibodies, peroxidase-labeled biotin-streptavid in, and 3,3'-diaminobenzidine tetra-hydrochloride (DAB) as the reveali ng system; 2) immunoperoxidase with tyramide signal amplification (TSA ), using biotinylated secondary antibodies, streptavidin-peroxidase, t yramide, streptavidin-peroxidase again and DAB; 3) indirect immunofluo rescence with fluorescein-labeled anti-mouse immunoglobulins. Results: The best results were obtained with the LSAB detection protocol prece ded by a predetection step with proteinase k. Proteinase k pretreatmen t did not significantly damage the tissue morphology and successfully unmasked beta 1 integrin antigens. Nonspecific background staining was reduced by a blocking step with swine serum. Similar results were obt ained with the TSA detection method; however, although lower concentra tions of anti-beta 1. integrin immunoglobulins and of secondary biotin ylated antibody were employed, there was more undesired background sta ining than with the LSAB protocol. Conclusions: The method reported an d discussed here may represent a valid tool for research and diagnosti c applications based upon detection of beta 1 integrin in paraffin-emb edded human tissues.