Nunc maxisorp 96 well plate

ACE-1 protein level was measured in mouse brain tissue homogenates using a commercially available mouse ACE-1 ELISA duoset following manufacturer’s guidelines (R&D Systems, Abington, Oxford, UK). In brief, a NUNC maxisorp 96-well plate (R&D Systems, Abington, Oxford, UK) was coated with a capture antibody (100μl) that was diluted in PBS (1600 ng/ml) and incubated overnight at room temperature. After washing (x5) and blocking for 1 h at room temperature with 1 % BSA:PBS, brain homogenates diluted in 1 % BSA:PBS (ACE-1: 1/80), a serial dilution of ACE-1 (125–8000 pg/ml), and 1 % BSA:PBS blanks were added to respective wells for 2 h at room temperature. After further washing (x5), the detection antibody diluted in 1 % BSA:PBS (400 ng/ml) was incubated for 2 h at room temperature. The plate was washed (x5) and streptavidin horse-radish peroxidase (1/200 in PBS:0.01 % Tween-20) was added for 20 min at room temperature in the dark. After a final wash step, TMB substrate (R&D Systems, Abington, Oxford, UK) was added to each well for 30 min at room temperature and 2 N sulphuric acid was added to stop the reaction. Absorbance was read at 450 nm for each well using a microplate reader (FLUOstar OPTIMA; BMG Labtech, Aylesbury, UK). ACE-1 concentration was interpolated from the serial dilution of recombinant mouse ACE-1. The average value for each sample was obtained from duplicates.

ACE-2 protein level was measured in brain tissue homogenates using a commercially available mouse ACE-2 ELISA duoset (R&D Systems, Abington, Oxford, UK) following manufacturer’s guidelines with minor modifications. Capture antibody was diluted at 4 µg/ml in PBS (2-fold higher than recommended) in a NUNC maxisorp 96-well plate (R&D Systems, Abington, Oxford, UK) and coated overnight at room temperature. After a wash and block step, brain tissue homogenates diluted 1 in 10 in PBS:1% BSA, a serial dilution of recombinant ACE-2, or a blank, was added to the wells in duplicate for 2 h at room temperature. Following a wash step, detection antibody at 200 ng/ml (2-fold higher than recommended) was added to the plate for 2 h at room temperature. Following a further wash step, streptavidin horse-radish peroxidase at 1 in 20 (2-fold higher than recommended) was incubated for 20 min. TMB substrate (R&D Systems, Abington, Oxford, UK) was added to each well for 30 min at room temperature and 2 N sulphuric acid was added to stop the reaction. Absorbance was read at 450 nm for each well using a microplate reader (FLUOstar OPTIMA; BMG Labtech, Aylesbury, UK). The concentration of ACE-2 was interpolated from the standard curve. Each sample was measured in duplicate and the average calculated.