Hpa012657

Renal PLA₂R of IMN patients was detected by indirect immunofluorescence in paraffin-embedded sections. Citrate buffer of pH 6.0 and microwaving at 100% power for 8 min were used for antigen retrieval and 3% bovine serum albumin was used for blocking. A commercial available anti-PLA₂R antibody (produced in rabbit, Sigma, HPA012657) was diluted at 1: 500 and incubated at 4 °C over-night. The secondary antibody was a fluorescein Cy3-conjugated donkey anti-rabbit IgG antibody (Chemicon, AP182C) and diluted at 1: 200. Each case was run with a positive and negative control (secondary antibody only) (Fig. 1).Fig. 1

Renal PLA₂R staining in IMN patients. (indirect immunofluorescence; original magnification ×400) a non-PLA₂R-associated IMN; b PLA₂R-associated IMN

For PLA₂R1 immunohistochemical analyses slides were deparaffinized, pre-treated in citrate buffer (pH 6.2) for 15 min at 120°C and cooled down in iced water (10 min). After rinsing in 99% ethanol, slides were incubated for 10 min with normal serum (Vector S2000; VectorLaboratories, Burlingame, CA) followed by PLA₂R1-antibodies (polyclonal antibody from rabbit, 1:3000, HPA 012657, Sigma-Aldrich, St. Louis, MO) overnight at 4°C. The slides were then washed in PBS, incubated with polymer 1 (Zytomed Zytochem-Plus AP Polymer-KitPOLAP), rinsed in PBS and incubated with polymer 2 (Zytomed Zytochem-Plus AP Polymer-Kit POLAP). After washing in PBS, slides were stained in new fuchsin naphthol As-Bi phosphate substrate mixture (30 min) followed by 1 min of nuclear staining in hemalaun (Mayer).