Gb21301

After deparaffinization and hydration, antigen recovery was performed with ethylene diamine tetraacetic acid antigen retrieval buffer (pH 8.0). Coronal sections were maintained at a subboiling temperature for 8 min to facilitate staining with an AQP4 antibody. Sections were then blocked in phosphate-buffered saline (PBS) with 3% bovine serum albumin (BSA) for 30 min, and stained sequentially with primary mouse antibody AQP4 (1:500, #GB12529, Servicebio) and Alexa Fluor 594 antimouse (1:300, #GB21301, Servicebio), together with the fluorescein isothiocyanate (FITC) conjugate Bandeiraea simplicifolia Isolectin B4 (BSI-B4, 1:50, #L-2895, Sigma). The brain sections were then covered with an antifade medium containing 4′,6-diamidino-2-phenylindole (DAPI, #D21490, Invitrogen). For this experimental step, IB4-FITC (2 mg dissolved in 2 ml saline, 0.05 mg per animal) was intravenously administered to the animals to identify the basement membrane. This was an important step as IB4 binds to a-D-galactose residues in the basement membrane secreted by endothelial cells (24 (link)). In each group, the circulation period of IB4-FITC was ensured for 180 min before sacrifice.

A549 cells were treated with 40 μM LY294002 or CA07 (MOI = 5) for 48 h. Cells were fixed with 4% pre-cooled formaldehyde for 15 min after removing the media, then followed by washing with PBS once and permeabilized with 0.1% Triton X-100 for 30 min. Next, the cells were blocked in PBS containing 5% bovine serum albumin (BSA) for 1 h at room temperature. After blocking, cells were incubated with anti-NP rabbit polyclonal (GTX125989, GeneTex) and anti-DNMT1 rabbit polyclonal (GTX116011, GeneTex) at 4 °C overnight. Cells were then washed with PBS and incubated with Alexa Fluoro 488-conjugated anti-rabbit antibody (ZF-0511, ZSJQ-BIO) and CY3 (GB21301, Servicebio) for 1 h at room temperature. Lastly, cells were stained by DAPI staining solution for 5 min. The images were captured using an Olympus Fluorescence IX73 Microscope.

Cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. Paraformaldehyde-fixed, paraffin-embedded tissue samples was cut into 4 μm slices and adhered to frosted glass slides. After washing with PBS and treating with PBS containing 0.1% Triton X-100 for 15 min, the sections and cells were permeabilized and blocked with 0.1% Tween-20 in PBS (PBST) containing 5% FBS for 90 min at room temperature. The cells were immunostained with anti-ACE2 (ab15348, 1:500, Abcam), anti-SP1 (T453) (ab59257, 1:500, Abcam), or anti-HNF4α antibodies (3113, 1:1 000, Cell Signaling Technology) overnight at 4 °C. The tissue sections were immunostained with anti-ACE2 (GB11267, 1:200, Servicebio, Wuhan, China) or anti-SARS-CoV-2-N antibodies (40143-MM05, 1:500, SinoBiological, Beijing, China) overnight at 4 °C. After washing three times with 0.1% Tween-20 in PBS (PBST), the cells were incubated with Alexa Fluor 594 anti-rabbit IgG (H+L) (A-21207, 1:200, ThermoFisher Scientific), Cy3 conjugated goat anti-mouse IgG (H+L) (GB21301, 1:300, Servicebio), or Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (GB25303, 1:500, Servicebio) at room temperature for 1 hr. After staining with primary antibodies, nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Images were acquired using a Zeiss Axioskop 2 plus fluorescence microscope (Carl Zeiss, Jena, Germany).

The xenograft tumors of the mouse model were fixed with formalin and embedded with paraffin. 4-μm sections were obtained from the paraffin-embedded tissues. After warmed at 55 °C for 30 min, the sections were dewaxed and rehydrated in xylene and ethanol, respectively. Then, the sections were boiled in sodium citrate-hydrochloric acid buffer solution for 10 min and blocked with 1% goat serum for 20 min at room temperature. The sections were incubated with mouse monoclonal antibody to MDA antibody (ab243066, Abcam, Cambridge, UK) at a dilution of 1:100 overnight at 4 °C, followed by incubation with a CY3-labeled goat anti-mouse IgG secondary antibody (GB21301, Servicebio) for 30 min at 37 °C. The cell nucleus was stained with DAPI.

The formalin‐fixed, paraffin‐embedded oral mucosal tissues were prepared, sectioned at 4 micrometres and analysed for the presence of LDHA antigen on the basis of standard immunohistochemistry protocols. Tissue sections were incubated overnight at 4℃ with LDHA rabbit polyclonal antibody (1:100, Catalog # 19987–1‐AP, Proteintech, Wuhan, China), followed by HRP polymer conjugated anti‐rabbit secondary antibody for 1 h at 37°C. Next, diaminobenzidine solution was used to detect the signal, and haematoxylin was used to counterstain the nucleus. Images were acquired using OLYMPUSCH30 microscope (BHS‐313, Olympus, Japan) at 200× and 400×. The mean optical density (MOD) was used to analyse the positive staining via Image‐Pro Plus 6.0 version (IPP, Media Cybernetics).
The sections for immunofluorescence staining were incubated with CD3 mouse monoclonal antibody (1: 100, Catalog # 60181–1‐Ig, Proteintech) and LDHA rabbit polyclonal antibody (1: 100, Proteintech) at 4°C overnight. Then, signals were observed using Cy3‐conjugated (1: 200, Catalog # GB21301, Servicebio, Wuhan, China) and Alexa Fluor® 488‐conjugated (1: 200, Catalog # GB25303, Servicebio) secondary antibodies. Nuclei were stained with DAPI. The slides were scanned under Pannoramic MIDI, and images were captured with CaseViewer (3DHISTECH). The fluorescence contrast parameters were described in detail in Appendix S3.