Gb25303

The sections were pretreated with 1% BSA in PBS containing 0.1% Triton X-100 for 1 hour, incubated in 1% Tween 20 for 20 min, and washed again with PBS. The sections were subsequently analyzed for Krt10 and Krt5, according to the manufacturers’ instructions. Sections were briefly incubated for 30 min in the dark, and excessive dye was rinsed off using PBS. Sections were then incubated with the antibody isotypes to exclude false-positive staining. Double immunofluorescence staining with primary antibodies against cytokeratin 10 (ab76318, Abcam, 1:150) and cytokeratin 5 (ab52635, Abcam, 1:200) and with secondary antibodies (GB25303 and GB21303, 1:400; Servicebio, Wuhan, China) was performed. The immunostained specimens were further subjected to Hoechst 33258 staining (G1011, Servicebio). At least three parallel sections were observed using ortho-fluorescent microscopy and imaging system (Nikon, Tokyo, Japan). Fluorescence area measurements were conducted at five random sites of regenerated epithelia using CaseViewer 2.1 and Image-Pro Plus 7.0 (n = 5).

Paraffin slides were de-waxed, rehydrated, subjected to heat-induced epitope retrieval (HIER), and followed by incubation with primary antibody to mouse CD8 (GB11068, Servicebio) or mouse CD4 (GB13064-2, Servicebio). Goat anti-rabbit Alexa Fluor 488 antibody (GB25303, Servicebio) was used as secondary antibody. DAPI (G1012, Servicebio) was used as the nuclear counterstain.

Immunofluorescence staining was performed on NRCMs grown on coverslips. NRCMs were fixed with 4 % paraformaldehyde for 15 min and blocked with 3 % BSA in PBS for 30 min at room temperature. For α-actinin staining, NRCMs were incubated at 4 °C for overnight with a rabbit anti-α-actinin antibody (GB111556, Servicebio, Wuhan, China) and a corresponding anti-rabbit Cy3 antibody (GB21302, Servicebio, Wuhan, China) for 1 hour at room temperature. The image was obtained via the fluorescence microscope. The surface area of NRCMs were depicted using Image J. For co-localization of USP38 and TBK1, NRCMs were incubated with rabbit anti-USP38 at 4 °C for overnight, then incubated with corresponding anti-rabbit HRP-labled antibody for 1 hour, followed by Cy3-Tyramide (GB1223, Servicebio, Wuhan, China) for 1 hour at room temperature. Subsequently, NRCMs were incubated with rabbit anti-TBK1 antibody at 4 °C for overnight, then incubated with anti-rabbit Alexa Fluro 488 antibody (GB25303, Servicebio, Wuhan, China) for 1 hour at room temperature. The nucleus was stained with DAPI (GB1012, Servicebio, Wuhan, China) for 10 min. The image was obtained via the confocal scanning microscope (NIKON Eclipse TI, Tokyo, Japan).

Fresh adipose tissues were obtained, fixed, and embedded in paraffin. After antigen repair and hydrogen peroxide blocking, the slides were blocked with a solution containing 3% BSA before incubation with primary antibodies at 4°C overnight. The slides were then treated with horseradish peroxidase (HRP)-conjugated anti-rabbit (Servicebio, GB23303, 1:500) or Alexa Fluor 488-conjugated anti-rabbit (Servicebio, GB25303, 1:400) secondary antibodies for 50 min at room temperature. The primary antibodies included CD44 (Servicebio, GB112054, 1:3000), CD62L (Bioss, bs-1036R, 1:1000), CD31 (Servicebio, GB113151, 1:1000), TH (Servicebio, GB11181, 1:1000), CD11B (Service bio, GB11058, 1:3000), CD115 (Servicebio, GB11581, 1:1000), and NK1.1 (Abcam, AB289542, 1:100). All the fluorescence pictures were captured by a fluorescence microscope (Nikon Eclipse C1) or laser scanning confocal microscope (Zeiss LSM 780).

The Olfr2 expression was performed by immunofluorescence (IF) analysis. Ana-1 cells or BMDMs were cultured on 14 mm round coverslips (BS-14-RC, Biosharp) in the 24-well plate (703001, Nest) and treated as described above. Cells were fixed with 4% PFA for 10min at RT. After washed three times with cold PBS, samples were incubated with 5% BSA (G1208, Servicebio) for 10min at RT and maintained in Olfr2 antibody (1:500, Thermo Fischer) at 4°C overnight. Subsequently, cells were washed three times and stained with secondary antibody (1:200, GB25303, Servicebio) for 1h at RT. DAPI (G1012, Servicebio) was used to stain nuclei in the dark for 10min. After washed three times, coverslips were sealed with anti-fluorescence quenching reagent (G1401, Servicebio) on glass slides. Images were captured by Zeiss scanning microscope and analyzed via ImageJ software.

For immunofluorescence double staining, freshly dissected bone tissues were fixed in 4% paraformaldehyde for 2 days and decalcified in 10% EDTA for 1 weeks. Slides were incubated with anti-CD105 (1:500, Abcam, ab107595), anti-CD200 (1:100, ProteinTech, 14057-1-AP), anti-Rabbit HRP (1:1000, Servicebio, GB23303), and anti-Rabbit 488 (1:1000, Servicebio, GB25303). Images were acquired with fluorescence inverted microscope (Leica, DMIL) and analyzed by ImageJ software.

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).