Anti fluorescence quenching agent

MLNs and spleen were excised and fixed in 4% paraformaldehyde. The tissues were then embedded in paraffin and sectioned at a thickness of 3–5 μm. After dewaxing, dehydration and antigenic reparation, the slices were stained overnight at 4 °C with primary antibodies against CD4, Foxp3, CD80, PD-L1 or CD11c (Servicebio, Wuhan, China) and incubated with HRP-conjugated or FITC-conjugated secondary antibodies (Servicebio, Wuhan, China) for 50 min at room temperature. Cell nuclei were labeled with DAPI for 5 min. The slices were sealed with an anti-fluorescence quenching agent (Servicebio, Wuhan, China) and imaged by fluorescence microscopy (Nikon, Tokyo, Japan).

The cells were inoculated on slides and grown to approximately 70% confluence. The slides were washed twice with phosphate-buffered saline (PBS) (Gibco, Waltham, MA, USA). The slides were fixed with 4% paraformaldehyde for 20 min, blocked with 3% BSA for 30 min, and incubated with primary antibodies against p-AMPK, collagen I, IL-8 (Cell Signaling Technology, Boston, MA, USA), vimentin, desmin, keratin, and S100 calcium binding solution (Thermo Fisher Scientific, Rockford, IL, USA) overnight at 4 °C. The slides were washed again and incubated with CY3-labeled goat anti-mouse secondary antibodies (Servicebio, Wuhan, China), goat anti-mouse IgG H&L (Alexa Fluor 647), and goat anti-rabbit IgG H&L (Alexa Fluor 488) (Thermo Fisher Scientific, Rockford, IL, USA) at room temperature in the dark for 1 h. Nuclei were stained for 5 min with DAPI (Servicebio, Wuhan, China). After a final wash, an anti-fluorescence quenching agent (Servicebio, Wuhan, China) was added to mount the slides, and images were collected with a microscope (Nikon, Tokyo, Japan).