Immunohistochemical Staining for ACE2 and TMPRSS2

Immunohistochemistry staining was performed as previous described.³⁰ (link) Briefly, deparaffinized sections were boiled in 100 × antigen retrieval buffer (ab93678, Abcam, Cambridge, MA) for 20 minutes. After blocking in 5% normal goat serum diluted in PBS, the sections were incubated with anti-ACE2 antibody (ab108252, 1: 100; Abcam, Cambridge, MA, USA), anti-TMPRSS2 antibody (ab92323, 1:2000; Abcam), and isotype- and concentration-matched normal IgG control (ab172730, Abcam) overnight at 4°C. The sections were incubated with secondary biotinylated goat anti-rabbit IgG (1:2000) for one hour at room temperature. ACE2 staining was then detected using the alkaline phosphatase detection system (Vectastain ABC-AP kit, Vector laboratories, Burlingame, CA), and a blue reaction product was produced by incubating sections with alkaline phosphatase substrate (Vector blue AP substrate kit III; Vector Laboratories, Burlingame, CA, USA) for 20 to 30 minutes. The sections were counterstained with nuclear fast red (Vector Laboratories), mounted with mounting medium (H-5501, Vector Laboratories), and examined under a light microscope. For quantitation of ACE2 positive blood vessels, figures at magnification ×10 were taken from four consecutive fields adjacent to the optic nerve. The number of ACE2 positive vessels was quantified by two independent observers in a masked fashion.