Prolong antifade

Immunofluorescence staining on PC-3 cells was performed essentially as described^{10 (link)}. Coverslips were mounted with Prolong antifade containing 4′,6′-diamino-2-phenylindole (DAPI) (Cell Signaling Technologies, Danvers, MA). Images were taken on a confocal microscope (LSM 510 Meta; Carl Zeiss, Inc.) using 63 × NA 1.40 Plan-Apochromat objectives and processed using Photoshop (Adobe).

Unstained paraffin-embedded kidney sections were treated with xylenes and alcohols to remove the paraffin. After unmasking the antigens, the sections were blocked with blocking buffer for 1 h and incubated overnight with primary antibodies (1:1000 dilution) against ACE (#SC12187), ACE-2 (#SC20998), ET-1 (#SC21625), ET-2 #(SC21627), HIF_1α (#SC10790), ET_AR, (#PIPA3065), ET_BR (#PIPA3066), from Santa Cruz Biotechnologies (Dallas, TX, USA), and Invitrogen (Suwanee, GA, USA), respectively. After washing with PBS, the sections were incubated with Cell Signaling (Danvers, MA, USA) Signal Stain Boost IHC reagent (HRP-conjugated), washed, mounted with Prolong Antifade, imaged and quantified using the CellSens software. Quantitative analysis was determined using the count and measure tool of the CellSens software (Olympus America, Center Valley, PA, USA). Images were captured at 20X magnification (1600 × 1200 pixels; scale bar = 50 μm). Resolution was enhanced using Adobe Photo Shop (San Jose, CA USA).