Mouse anti p rr

Renal cortical tissues were obtained from normotensive IgAN patients (eight from patients with proteinuria <1 g/day and another eight from patients with proteinuria >1 g/day). They had not previously received angiotensin-converting enzyme inhibitor or AT1R antagonist. Control renal tissues were obtained from the intact pole of kidneys removed for single circumscribed tumor in seven normotensive subjects. Paraffin-embedded renal tissues were sectioned at a thickness of 5 μm. The sections were deparaffinized with xylene and then rehydrated through a descending gradient of ethanol. Glomerular expression of TNF-α, TNFR1, AT1R, PRR and CTGF were detected by immunoperoxidase staining using specific rabbit or mouse antibodies (rabbit anti-TNF-α, TNFR1, AT1R, CTGF from Abcam; mouse anti-PRR from Sigma; final dilution at 10 μg/ml). The bound rabbit or mouse antibodies were visualized in brown colour using the Dako Envision Plus System (Dako). Apoptosis was determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining carried out with the ApopTaq Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore, Billerica, MA, USA) following the manufacturer’s protocol. Cells were regarded as apoptotic if they exhibited brown stained nuclei.