Low fluorescent pvdf membrane

The kidney cortex was crushed in liquid nitrogen with a mortar and pestle and then homogenised with protein extraction buffer as described previously [20 ]. Protein concentration was established using the DC Protein Assay Kit. Expression of AGTR1 and AGTR2 proteins was then measured using the Amersham ECL Plex Western blotting system using low-fluorescent PVDF membrane (GE Healthcare, Buckinghamshire, UK) as described previously [20 ]. Briefly, anti-AGTR1 ab18801 and anti-AGRT2 ab19134 antibodies were used. We have reported the optimisation of anti-AGRT2 ab19134 [20 ]. Anti-histone H2B (ab52484) primary antibody at 1:40000 dilution incubation with membrane for 1 h at room temperature was followed by washing and then incubation with secondary antibody as described previously [20 ]. Signal on the membrane was detected by fluorescent laser scanner (Typhoon, GE Healthcare, Buckinghamshire, UK), and images were quantified using ImageQuant (GE Healthcare). The optimal primary and secondary antibody concentrations were tested. Specificity of AGTR1 (Additional file 1: Figure S1) and AGTR2 [20 ] antibodies was confirmed by blocking peptide: AGTR1 -ab91523 (Abcam, Cambridge, UK) and AGTR2 -ab91522 (Abcam). Expression of AGTR1 and AGTR2 was normalised to histone H2B expression, which did not vary between males and females, with age or surgical treatment.