Sa00003 3

Brain tissues were fixed, dehydrated, and then cut into coronal sections at a thickness of 10 μm. Sections were permeabilized with 0.3% Triton X-100. Then, brain slices were blocked with 5% donkey or goat serum for 1 h and incubated overnight at 4°C with primary antibodies: ABIN1 (bs-9568R, Bioss, China, 1 : 50), NeuN (MAB377, Millipore, Germany, 1 : 200), A20 (3A11G6, Proteintech, USA, 1 : 100), Iba-1 (NB100-1028, Novus, USA, 1 : 50), and GFAP (BM0055, Boster, China, 1 : 100). Then, the following fluorescently labeled secondary antibodies were incubated with the sections at 37°C for 1 h in the dark: CoraLite594-goat anti-rabbit (SA00013-4, Proteintech, 1 : 200), CoraLite488-goat anti-mouse (SA00013-1, Proteintech, 1 : 200), CoraLite594-donkey anti-rabbit (SA00013-8, Proteintech, 1 : 200), FITC-donkey anti-goat (SA00003-3, Proteintech, 1 : 200), and Cell nuclei were stained with DAPI. Brain slices were observed under a laser confocal microscope (LSM-800, Carl Zeiss Micro-Imaging Co., Germany).

Paraffin-embedded sections obtained from the mouse metastatic lung or human metastatic lung were subjected to immunofluorescence (IF) staining according to a standard immunofluorescence paraffin-embedded tissue staining protocol. In brief, after lung tissue sections were dewaxed in xylene and rehydrated, antigen retrieval was carried out in 10 mM sodium citrate (pH 6.0) at 120 °C for 5 min using a pressure cooker. The tissue sections were then blocked with 5% normal goat serum for 1 h at room temperature and further incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. The following antibodies were used for staining: Ly6G (Biolegend, catalog no. 127602), MPO (R&D Systems, catalog no. AF3667), and MHCII (Invitrogen, catalog no. 14-5321-82). The tissue sections were subsequently washed with PBS and incubated with fluorophore-labeled, appropriate secondary antibodies for 1 h at room temperature. The secondary fluorescent antibodies used included donkey anti-goat (Proteintech, catalog no. SA00003-3) and donkey anti-rat (Proteintech, catalog no. SA00007-7) antibodies. DAPI (Vector Laboratories, catalog no. H-2000) was used to mark the nuclei, and fluorescence images were captured by a Nikon confocal A1 laser microscope (Nikon). All image quantification was performed using ImageJ software (version 1.8.0).

Immunofluorescence was performed by a method described previously [29 (link)]. After anesthesia, 0.9% saline and 4% formaldehyde were transcardially infused into rats. The brains were carefully removed and dehydrated by 15% and 30% sucrose. Rats were anaesthetized and transcardially perfused with 0.9% saline and 4% formaldehyde. The brains were removed carefully and dehydrated with 15% sucrose and 30% sucrose. Ten-micrometer-thick coronal brain sections were harvested and incubated with 1% Triton X-100 for 30 min at room temperature. Subsequently, the sections were blocked for 1 h at 37°C with 5% bovine serum albumin and then incubated with anti-GFAP mouse antibody (A00213, BOSTER Co.) and anti-Iba-1 goat antibody (NB100-1028, Novus Co., USA, 1 : 200) at 4°C overnight. The next day, the sections were incubated with FITC-conjugated AffiniPure donkey anti-goat IgG (H+L; SA00003-3, Proteintech, 1 : 200) or Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L; SA00006-1, Proteintech, 1 : 200). The DAPI was used to stain nuclei (Sigma, USA, 1 : 200). The images were taken using an A1+R laser confocal microscope (Nikon, Tokyo, Japan).

The slides of brain sections were fixed with 4% formaldehyde solution for 30 min at room temperature, incubated with 1% Triton X-100 for 30 min, and blocked with 5% goat or donkey serum for 1 h at 37 °C. Subsequently, sections were incubated overnight at 4 °C with the following primary antibodies: OTULIN (bs-14689R, Bioss Co., Beijing, China, 1:50), NeuN (MAB377, Millipore Co., Germany, 1:200), MAP2 (4542, Cell Signaling Technology, USA, 1:100) or Iba-1 (NB100-1028, Novus Co., USA, 1:200). After washing three times with PBS, sections were reacted with the following fluorescent secondary antibodies at 37 °C for 1 h: Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L; SA00006-4, Proteintech, 1:200), Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L; SA00006-1, Proteintech, 1:200), FITC-conjugated AffiniPure donkey anti-goat IgG (H+L; SA00003-3, Proteintech, 1:200), and 594-conjugated AffiniPure donkey anti-rabbit IgG (H+L; SA00006-8, Proteintech, 1:200). DAPI (Sigma, USA, 1:200) was used to stain cellular nuclei at 37 °C for 10 min. All images were observed and acquired using an A1+R laser confocal microscope (Nikon, Tokyo, Japan).

Flow cytometry was performed to verify the binding of SARS-CoV-2 Spike protein and human ACE2 protein in vitro. In brief, HEK293T cells were transfected with either empty vector or ACE2 plasmid for 48 h. Then, cells were incubated with SARS-CoV-2 spike RBD-Fc recombinant protein (10 µg) or control-Fc protein (10 µg) in PBS buffer at 37℃ for 2 h, then followed by incubation with anti-Human IgG (Fc specific) antibody (I2136-1ML, Sigma, 1:1000) and Donkey Anti-Goat IgG(H + L), FITC conjugate antibody (1:1000; SA00003-3, Proteintech). Native HEK293T cells incubated with SARS-CoV-2 spike RBD-Fc followed by anti-Fc antibody were shown as controls. After washing cells with PBS, the cells were analyzed by CytoFLEX Flow Cytometry (CytoFLEX S, BECKMAN COULTER). In brief, the live cells were gated according to the values of forward scatter (FSC) and side scatter (SSC). Then, the values of FL1 channel were recorded to determine the intensity of Human IgG (Fc)-FITC. The results were analyzed by FlowJo software.