Aims and background: Integrins are widely known cell membrane receptors for extracellular matrix molecules. The beta 1 integrin subgroup is mainly expressed by kidney cells; immunolocalization of these molecules is usually carried out on cryostatic sections. A commercial monoclonal antibody directed against the human beta 1 integrin was tested in order to design a method for the detection of this antigen in formalin-fixed, paraffin-embedded human kidney tissue. Methods: Specimens obtained from nephrectomies were fixed with 10% formalin and embedded in paraffin. Three different detection protocols were applied after incubation with the antihuman beta 1 integrin monoclonal antibody (MAB 1977): 1) immunoperoxidase with labelled streptavidin biotin (LSAB), using biotinylated secondary antibodies, peroxidase-labeled biotin-streptavidin, and 3,3'-diaminobenzidine tetra-hydrochloride (DAB) as the revealing system; 2) immunoperoxidase with tyramide signal amplification (TSA), using biotinylated secondary antibodies, streptavidin-peroxidase, tyramide, streptavidin-peroxidase again and DAB; 3) indirect immunofluorescence with fluorescein-labeled anti-mouse immunoglobulins. Results: The best results were obtained with the LSAB detection protocol preceded by a predetection step with proteinase k. Proteinase k pretreatment 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 obtained with the TSA detection method; however, although lower concentrations of anti-beta 1. integrin immunoglobulins and of secondary biotinylated antibody were employed, there was more undesired background staining than with the LSAB protocol. Conclusions: The method reported and discussed here may represent a valid tool for research and diagnostic applications based upon detection of beta 1 integrin in paraffin-embedded human tissues.
The use of MAB 1977 monoclonal antibody for the immunohistochemical localization of beta 1 integrins in paraffin-embedded human kidney
BERNARDINI, NUNZIA;BIANCHI, FRANCESCO;LUPETTI, MARIO;DOLFI, AMELIO
1997-01-01
Abstract
Aims and background: Integrins are widely known cell membrane receptors for extracellular matrix molecules. The beta 1 integrin subgroup is mainly expressed by kidney cells; immunolocalization of these molecules is usually carried out on cryostatic sections. A commercial monoclonal antibody directed against the human beta 1 integrin was tested in order to design a method for the detection of this antigen in formalin-fixed, paraffin-embedded human kidney tissue. Methods: Specimens obtained from nephrectomies were fixed with 10% formalin and embedded in paraffin. Three different detection protocols were applied after incubation with the antihuman beta 1 integrin monoclonal antibody (MAB 1977): 1) immunoperoxidase with labelled streptavidin biotin (LSAB), using biotinylated secondary antibodies, peroxidase-labeled biotin-streptavidin, and 3,3'-diaminobenzidine tetra-hydrochloride (DAB) as the revealing system; 2) immunoperoxidase with tyramide signal amplification (TSA), using biotinylated secondary antibodies, streptavidin-peroxidase, tyramide, streptavidin-peroxidase again and DAB; 3) indirect immunofluorescence with fluorescein-labeled anti-mouse immunoglobulins. Results: The best results were obtained with the LSAB detection protocol preceded by a predetection step with proteinase k. Proteinase k pretreatment 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 obtained with the TSA detection method; however, although lower concentrations of anti-beta 1. integrin immunoglobulins and of secondary biotinylated antibody were employed, there was more undesired background staining than with the LSAB protocol. Conclusions: The method reported and discussed here may represent a valid tool for research and diagnostic applications based upon detection of beta 1 integrin in paraffin-embedded human tissues.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
