PLA2R1 Antibody (12-6-5):
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
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 um) 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.
