Fitc labeled goat anti mouse igg

Infected and mock-infected cells were harvested at 24 h postinfection and immediately fixed on slides with 4% paraformaldehyde for 30 min at room temperature. The fixed cells were washed with PBS and permeabilized with 0.5% Triton X-100. The cells were then inoculated with a mouse anti-influenza NP antibody (Abcam, UK) at 4°C overnight, followed by washing with PBS and incubation with FITC-labeled goat anti-mouse IgG (Abcam, UK) for 30 min at room temperature in the dark. DNA staining was performed using DAPI mounting medium. The slides were then washed again and air-dried, and images were captured using a Nikon Eclipse Ti-S fluorescence microscope.

After treatment, lung tissues were collected. Paraffin embeded lung tissues were cut into slices for histological staining. HE staining was performed according to the manufacturer’s instructions (Solarbio). The mean alveolar septal thickness (MAST), mean linear intercept (MLI), and destructive index (DI) were calculated. Immunohistochemistry staining was conducted using primary antibodies for SLC27A3 (Proteintech), STAU1 (Affinity), CD57 (Affinity), and CD8a (Affinity). Immunofluorescence staining was performed using primary antibodies for CD19 (Affinity) and CD27 (Santa), and secondary antibodies including Cy3-labeled goat anti-rabbit IgG (Invitrogen) and FITC-labeled goat anti-mouse IgG (Abcam).

DC 2.4 cells were seeded in 24-well plates at 500,000 cells/well and then transfected with 1 μg of mRNA to form cell monolayers using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific). After 6 h, the medium was replaced with DMEM supplemented with 3% FBS. Following 24 h post-transfection, cells were fixed with ice-cold acetone for 10 min and incubated with a polyclonal mouse anti-CHIKV antibody (prepared in our laboratory) and then FITC-labeled goat anti-mouse IgG (Abcam). Nuclei were counterstained with DAPI (Thermo Fisher Scientific). Fluorescence was observed using a fluorescent microscope (Olympus).
After 16–26 h post-transfection, cells were lysed with RIPA buffer plus proteinase inhibitor (Sigma), clarified by centrifugation at 12,000 × g, and then 5 × SDS loading buffer was added, and cells were kept at 95°C for 10 min. The lysates were run on a 10% NuPAGE Bis-Tris gels (Invitrogen) followed by transferring proteins to a nitrocellulose membrane. The membrane was incubated with a polyclonal mouse anti-CHIKV antibody (prepared in our laboratory) and HRP-labeled goat anti-mouse IgG (Abcam). DC 2.4 cells with lysing buffer and E2 protein were used as negative and positive controls (ProSpec), respectively. Blots were developed using ECL reagents (GE).

Cx43-hUCSCs were cultured on glass cover slips in six well plates and grown to 80% confluence. Cells were washed with PBS and fixed with 40 mg/L paraformaldehyde (Sigma, USA) for 25 min, then blocked with 10% goat serum albumin (Sigma, USA) for 20 min before staining. A drop of DAPI staining solution (Lifetechnologies, USA, 1 mg/ml) was added to stain the cells for 5 min. The slips of cells were washed with PBS, and a drop of 1:100 diluted mouse anti-human Cx43 monoclonal antibody (Abcam, USA) was added and incubated for 24 h at 4°C. Samples were warmed to room temperature for 1 h, washed with PBS, 1 drop of 1:100 diluted FITC-labeled goat anti-mouse IgG (Abcam, USA) was added and incubated for 1 h at room temperature. Mouse monoclonal IgG1 (Abcam, USA) was used as isotype control as instructed by manufactures. After washing with PBS, the slips were sealed using 60% buffering glycerol (Sigma, USA) and imaged by a laser confocal microscope (Leica, Germany) with a fast scanning mode.

The lung tissue slides embedded in paraffin were exposed to citrate solution for antigen retrieval after dewaxing and rehydration. The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 15 min and incubated with MPO antibody (1:100 dilution; ABclonal, Shanghai, China) or P65 antibody (1:100 dilution; ABclonal, Shanghai, China) overnight at 4 °C. For double immunofluorescence staining, the slides were probed with PTPRO (1:50 dilution, Santa, USA) and keratin 19 (1:100 dilution; ABclonal, Shanghai, China) or beta IV Tublin (1:100 dilution; Affinity, Cincinnati, OH, USA). Then the tissues were incubated in the dark with a Cy3-conjugated goat anti-rabbit IgG (1:200 dilution; Beyotime, Shanghai, China) or FITC-labeled goat anti-mouse IgG (1:200 dilution; Abcam, Cambridge, UK) at room temperature for 1 h. The tissues were re-stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China), and the images were captured at 400 × magnification by a fluorescence microscope (BX53, Olympus, Tokyo, Japan).