Vectastain elite abc avidin biotin complex kit

FFPE tissue was cut into 2 μm sections on a microtome and de-paraffinized. After antigen retrieval slides were incubated with primary antibody (the primary antibodies used are listed in Table S3) over night at 4 °C and washed with Tris-HCL (Tris hydrochloride) buffer (pH 7.5) followed by a secondary antibody. Antibodies were detected with the Vectastain Elite ABC (avidin-biotin complex) kit (Vector) using DAB (3,3′-diaminobenzidine) (Vector Laboratories and Dako) for brown stainings or AEC (3-Amino-9-ethylcarbazole) (Thermo Fisher Scientific) for magenta stainings. The slides were counterstained with hematoxylin (Vector Laboratories) and mounted with Roti®-Histokitt II (Carl Roth, Germany). Images were captured on an Axioplan2 imaging microscope (Carl Zeiss) equipped with an AxioCamHRc Camera (Carl Zeiss) or slides were scanned with a Vectra Polaris imaging system (PerkinElmer, Hopkinton, MA, USA) and quantified by ImageJ software.

Formalin-fixed, paraffin-embedded tissue sections (7 μm) were cut and de-waxed prior to immunohistochemistry. Sections were pre-treated in trisodium citrate buffer (9 mM), pH 6, and microwaved for 5 minutes, left to stand for 5 minutes, and boiled for a further 5 minutes before being left to stand for 15 minutes at room temperature. Sections were then rinsed thoroughly and covered in horse serum blocking solution, rinsed again, and incubated overnight at room temperature with anti-ACE-2 antibody (0.05 μg/ml, ab15348; Abcam). Bound antibody was visualised using a biotinylated universal antibody followed by VECTASTAIN Elite ABC avidin-biotin complex kit (Vector Laboratories, Peterborough, UK) and a reaction with 0.01% H₂O₂. Specificity of the antibody was assessed by pre-adsorption of the ACE-2 antibody with a 250-fold molar excess of recombinant human ACE-2 protein (R&D Systems).