The use of MAB 1977 monoclonal antibody for the immunohistochemical localization of beta 1 integrins in paraffin-embedded human kidney.
OBJECTIVE
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 anti-human beta 1 integrin monoclonal antibody (MAB 1977): 1) immunoperoxidase with labeled 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 beat 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.
Bernardini
N
N
Cattedra di Istologia ed Embriologia, Istituto di Anatomia Umana Normale, Facoltà di Medicina e Chirurgia, Università di Pisa, Italy.
Bianchi
F
F
Lupetti
M
M
Dolfi
A
A
D018262:Adenocarcinoma, Clear Cell / Q000276:immunology; D000911:Antibodies, Monoclonal; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007124:Immunoenzyme Techniques; D019012:Integrin beta1 / Q000032:analysis*; D007668:Kidney / Q000276:immunology*; D007680:Kidney Neoplasms / Q000276:immunology*; D009392:Nephrectomy; D010232:Paraffin
D000911:Antibodies, Monoclonal; D019012:Integrin beta1; D010232:Paraffin