Bs114

Different concentrations of COF samples were added to 4T1 cells and incubated overnight. Following various treatments, the 4T1 cells (2 × 10³) were fixed with 4% paraformaldehyde, infiltrated with 0.5% Triton X-100 (BS084, Biosharp), blocked with 3% BSA (Biosharp BS114) in 0.1% triton/PBS for 1 hour, and incubated with anti-HMGB1 antibodies (1:400, ab18256, Abcam) at room temperature and Goat anti-Rabbit IgG DyLight 488 (1:200, A23220, Abbkine). The cells were then counterstained with DAPI (Beyotime Biotechnology, P0131), The slides were examined with an FV1000 confocal microscope (Olympus) using FV10-ASW software v. 4.0 (Olympus). The fluorescence signals were analyzed using Image-Pro Plus software v. 6.0 (Media Cybernetics).

The localization of PERK, STING, p-TBK1, and p-IRF3 were identified by immunofluorescence. Paraffin sections of brain tissue were dewaxed with xylene and dehydrated with gradient alcohol. Antigen repair was performed with EDTA (pH 8) and heated in the microwave. After cooling naturally, the sections were washed thrice with phosphate-buffered saline (PBS, pH 7.4) and blocked using 5% bovine serum albumin (BSA; #BS114, Biosharp) for 1 h at room temperature. Subsequently, slices were incubated overnight at 4°C with mouse anti-GFAP (glial fibrillary acidic protein, #GB12096,1:500, Servicebio) to identify astrocytes, mouse anti-ionized calcium-binding adaptor molecule 1 (Iba-1; #GB12105,1:500, Servicebio) to identify microglia, mouse anti-NeuN (Neuronal Nuclei, #K009907M,1:200, solarbio) and primary antibody against PERK (1:50), STING (1:100), p-TBK1(1:100), and p-IRF3 (1:200), respectively. After three washes with PBS on the next day, slices were incubated in Cy3-conjugated anti-rabbit (GB21303,1:200, Servicebio) and AlexaFluor488-conjugated anti-mouse (GB25301,1:400, Servicebio) secondary antibodies for 50 min in the dark. Finally, DAPI (G1012, Servicebio) was added to the sections for 15 min to identify the nuclei. Images were acquired on an inverted fluorescence microscope (Eclipse C1; Nikon) at 40× objective and an imaging system (DS-U3; Nikon).