Anti ace2

For immunofluorescence double-staining, the sections were incubated with primary mouse originated antibodies including anti-SARS NP antibodies (ab273434, 1:500, mouse IgG), anti-SARS S antibodies (ab273433, 1:500, mouse IgG), or anti-DP2 (sc-271898, 1:200, mouse monoclonal C-5; Santa Cruz Biotechnology) and rabbit originated antibodies including anti-SARS NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological) or anti-PDS (ab182141, 1:100, rabbit IgG1; Abcam) at 4 °C overnight, respectively. After washing with PBS (3 washes, 5 min per wash), the sections were incubated with Alexa Fluor^® 555-conjugated goat anti-rabbit IgG antibodies (Invitrogen, San Diego, CA, USA) and Alexa Fluor^® 488-conjugated goat anti-rabbit IgG1 antibodies (Invitrogen) for an 1 h. Finally, the sections were incubated with 1 μg/ml DAPI (Sigma, St. Louis, MO, USA) for 10 min to stain the nuclei. Sections incubated with the appropriate isotype control primary antibodies and fluorescently labeled secondary antibodies were used as negative controls. The results were analyzed using fluorescence microscopy (Zeiss Axioplan 2).

Multicolor immunofluorescence analyses were performed using SARS-CoV-2 infected cells and lung tissues from patient with COVID-19. In brief, slides were dewaxed, followed by antigen retrieval in citrate buffer. Then the samples were permeabilized with 0.5% TritonX-100. After blocked with 5% goat serum, the slide was sequentially incubated with anti-spike (40150-R007, Sino Biological, China), anti-CD147 (Jiangsu Pacific Meinuoke Biopharmceutical Co. Ltd, China), anti-ACE2 (80031-R003, Sino Biological, China), anti-Rab5 (3547s, Cell Signaling Technology, USA), and anti-CD3 (ab5690, Abcam, UK) antibodies. TSA-Indirect Kit (PerkinElmer, USA) was used according to the manufacturer’s manual. Images were analyzed with Heilo software.

The protocol for immunohistochemistry has been reported in our previous work^{45 (link)}. Briefly, antigen retrieval was performed by microwaving these sections in citrate buffer (10 mM, pH 6.0). The sections were then incubated in 3% BSA plus 0.1% H₂O₂ for 1 h at RT to block nonspecific binding. The sections were then incubated overnight at 4 °C with primary anti-SARS-CoV-2 NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-SARS-CoV-2 NP antibodies (ab273434, 1:500, mouse monoclonal 6H3, Abcam), anti-SARS spike glycoprotein (S) antibodies (ab273433, 1:500, mouse monoclonal 1A9, Abcam), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological), anti-CD8 (Clone ID:4B11, 1:100, mouse IgG2b; BIO-RAD), anti-CD68 (Clone ID:KP1, 1:100, mouse IgG1; BIO-RAD), anti-CD56 (Clone ID:123C3, 1:100, mouse IgG1; BIO-RAD), anti-C5b-9 (clone ID: aE11, 1:100, mouse IgG; Dako cytomation). Sections were further incubated with the Goat anti-Rabbit IgG (H + L) secondary antibody, HRP (#31460, Invitrogen) or Goat anti-Mouse secondary antibody, HRP (PA1-74421, ThermoFisher) for 1 h at RT, respectively. Peroxidase activity was visualized with the DAB Elite kit (K3465, DAKO), and the brown coloration of tissues represented positive staining as viewed by a light microscope (Zeiss Axioplan 2, Germany).