Ab9391

Five micron thick FFPE sections affixed to slides were de-waxed and antigen retrieval
for prorenin, ATP6AP2, ACE1, ACE2, AGT, AGTR1 and MAS1 was performed in 10 mM
citrate buffer at pH 6.0 for 10 min using a microwave oven. Antigen retrieval
for AGTR2 was performed in a hybridisation oven with Proteinase K (1:1000) for
10 min at 37°C for 10 min. Antibodies used were ACE1 (BosterBio,
PA2196-1, 2.5 μg/mL), ACE2 (Abcam, ab15348, 0.005 mg/mL), AGT
(R&D Systems, af3156, 0.002 mg/mL), AGTR1 (Abcam, ab9391,
0.125 mg/mL), AGTR2 (Abcam, ab19134, 0.012 mg/mL), MAS1 (Abcam,
ab140854, 0.1 mg/mL), ATP6AP2 (Everest Biotech, eb06118,
10 μg/mL) and REN propeptide (R&D Systems, MAB4447,
0.05 mg/mL). The positive control tissue was a first trimester placenta sample
collected at elective termination of pregnancy. Matched samples without the addition
of primary antibody were included as negative controls. Sections were blocked with
bovine serum albumin (BSA) blocking solution (0.5% BSA w/v, 0.05% w/v Saponin in
0.1 M PBS) for 1 h at room temperature and then incubated overnight
with primary antibody. Images were captured using the Aperio AT Turbo slide scanner
(Leica Biosystems).

After fixation, the renal tissues were embedded in paraffin and sectioned at 3-µm thickness. Following dewaxing via a hydration process, antigen repair was performed using the high-pressure hot-repair method, and endogenous antigen was inactivated using peroxidase. The sections were then washed in phosphate-buffered saline (PBS), incubated in goat serum working fluid for 30 min, and then separately with the following primary antibodies. All antibodies were purchased from Abcam (Cambridge, UK; anti-AT1R antibody: ab9391, anti-AT2R antibody: ab19134, anti-MAS1 antibody: ab66030). After an overnight incubation at 4°C, the reactions were visualized by staining with horseradish peroxidase and diaminobenzidine.
The protein expression of AT1, AT2, and MAS receptors was examined quantitatively using the Image-Pro Plus (IPP) 6.0 software program (Media Cybernetics, Silver Spring, MD, USA). First, we select the area of interest (AOI) by setting the color range (yellow or brown), and then measure the average optical density in the AOI. The mean density of the AOI was used to reflect the relative content of the target protein. A mean value was obtained by analysis of 10 different fields. Quantification was done twice in a blinded manner, and interassay variations were not significant.