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|Applications||FACS IHC IF WB|
|Antigen/Gene or Protein Targets||Phospholipase A2 receptor 1|
|Relevance||PLA2R is the major auto-antigen in the autoimmune kidney disease, membranous nephropathy. Detection of PLA2R antigen by IHC in the immune complexes present in the glomerular basement membrane is used as a diagnostic marker for the disease. PLA2R acts a tumour suppressor protein and it is associated with cell senescence and differentiated cells.|
|Immunogen||Human PLA2R extracellular sequence N-C3 (containing domains N-terminal Cysteine rich-FibII-CTLD1-CTLD2-CTLD3). Genbank acc no. U17033; amino acids 20-663 Recombinant protein expressed in HEK 293-EBNA-1 cells|
KD 5.4x10-10 M (determined by surface plasmon resonance between recombinant human PLA2R and increasing concentration of Ms Clone 12-6-5 antibody).
System: Automated Ventana Benchmark ULTRA
Detection Kit: UltraView DAB Detection System
Antigen Retrieval: 64 minute heat in CC1 (Tris based pH8.4 buffer)
Antibody concentration: 1+5000
Antibody Incubation: 36 minute at room temperature.
Counterstain: Haematoxylin 12 min, Bluing Reagent 4 min.
Positive Tissue Control: Normal kidney
Extended information including detection system reagents: The automated Ventana BenchMark ULTRA IHC ⁄ ISH Staining Module (Ventana Co., Tucson, AZ, USA) was used together with the Ultraview 3, 3’ diaminobenzidine (DAB) version 3 detection system (Ventana Co.). Tissue sections (4 µm) were deparaffinized and incubated in EZPrep Volume Adjust (Ventana Co.). At intervals between steps the slides were washed with a TRIS-based Reaction Buffer, pH 7.6. A heat-induced antigen retrieval protocol set for 64 min was carried out using a TRIS– ethylenediamine tetracetic acid (EDTA)–boric acid pH 8.4 buffer (Cell Conditioner 1). The sections were incubated with ultraviolet inhibitor blocking solution for 4 min, then with antibody to PLA2R for a set time of 36 min at room temperature. This was followed by incubation with horseradish peroxidase-linked secondary antibody (8 min.), followed by DAB chromogen and substrate (8 min.), and copper enhancer for 4 min. Counterstain (haematoxylin II) was applied for 12 min before an incubation of 4 min with bluing reagent.
|Research Area||Cardiovascular, Cell Signaling & Signal Transduction, Metabolism|
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