As previously described [21], cells were fixed in 4% paraformaldehyde for 30 min and washed with phosphate-buffered saline (PBS), followed by permeabilization in 0.2% Triton X-100 for 20 min. Samples were blocked with 5% bovine serum albumin (ST023; Beyotime, Shanghai, China) for 1 h and then incubated with primary antibodies against GFAP (MAB360; Merck Millipore, Billerica, MA, USA), p65 (8242; Cell Signaling, Boston, MA, USA), C3 (EPR9394; Abcam, Cambridge, UK), or S100A10 (11250-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C overnight. Following washes with PBS, samples were incubated with Alexa Fluor anti-mouse (A11001) or anti-rabbit IgG (A21428) at room temperature for 1 h, and subsequently incubated with Hoechst 33258 (H3569; all these three from Thermo, Rockford, IL, USA) for 5 min. Coverslips were mounted on glass slides via Fluoromount (P0126; Beyotime, Shanghai, China), and observed with Nikon Eclipse Ti-S.

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