Dapi c1006

FaDu and SCC15 cells seeded on sterile coverslips were fixated with 4% paraformaldehyde for the duration of 20 min, then permeabilized with 0.1% Triton X-100 for 15 min, and lastly blocked with goat serum at room temperature for a total of 30 min. Subsequently, primary antibodies were utilized for incubation of the cells overnight at 4 °C, of which the details are shown in Additional file 2: Table S1. The following day, fluorescence-labeled secondary antibodies were utilized incubation of the cells for 1 h at room temperature. DAPI (C1006, Beyotime, Shanghai, China) was used to stain nuclear DNA and the images were captured with a confocal system (Nikon).

The immunofluorescent process of tissue or cells was done as previously described.40, 43 The detailed information of primary and secondary antibodies was shown as following: NF‐200 (1:10000, Abcam, ab4680), MBP (1:200, Abcam, ab40390), HO‐1 (1:100, Santa Cruz, sc‐1797), NQO1 (1:1000, Abcam, ab34173), S100 (1:200, Abcam, ab4066), Nrf‐2 (1:500, Abcam, ab62352), Alexa‐Fluor 488 donkey anti‐ rabbit IgG (1:1000, Abcam, ab150073), and Alexa‐Fluor 594 donkey anti‐mouse IgG (1:1000, Abcam, ab150108). Nuclei were labelled with 4′6‐Diamidino‐2‐phenylindole‐dihydrochloride (DAPI, C1006; Beyotime Institute of Biotechnology, Shanghai, China). All fluorescence images were obtained under the Nikon ECLIPSE 80i (Nikon, Tokyo, Japan).

After the induction of M1 polarization in RAW 264.7 cells, the expression of M1 marker CD63 was detected by IF. Briefly, cells were fixed with 4% paraformaldehyde for 30 min at 25 °C and then permeabilized by 0.5% TritonX100 for 15 min at 25 °C. After washing three times with PBS, cells were incubated in a blocking solution of 3% BSA (diluted in PBS) for 30 min at 25 °C. Next, cells were incubated with pre-cooled anti-CD63 (1:200, ab239075; Abcam) at 4 °C overnight followed by incubation with goat anti-rat Alexa Flour 488 (1:300, ab150077; Abcam). Finally, cells were stained with DAPI (C1006; Beyotime, Shanghai, China) for 10 min in the dark condition and observed the fluorescence on a fluorescence microscope.

Mice were carefully anesthetized at 7 days after MCAO and then rapidly perfused with 0.9% sodium chloride. The brains was immediately removed and dehydrated with PBS containing 30% sucrose for 12 h. Afterwards, a frozen section slicer (Cryotome E, Thermo) was used to section the samples, which were then embedded and frozen in OCT-Freeze medium (4583, Sakura). Cryosections were incubated with 10% goat serum for 2 h at room temperature to block non-specific binding of immunoglobulin. Following blocking, the sections were incubated with rabbit anti-GluA2 antibodies (1:400, MAB397, Merck Millipore) at 4°C overnight as the primary antibody. After washing in PBS, the sections were incubated with the appropriate secondary antibody for 30 min at 37°C (DyLight 488 affiniPure goat anti-rabbit IgG, 1:100, A23220, Abbkine, USA). Synapses were determined via incubation with an antibody against synaptophysin (1:500, ab130436, Abcam). Subsequently, the slices were incubated with the secondary antibody (Alexa-594, A-11032) for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, C1006, Beyotime, China). Images were captured with a fluorescence microscope (Eclipse Ti-S, Nikon, Japan), and visual fields in each section were analyzed using Image-pro plus (IPP) 6.0 software.

Tissue specimens were blocked in 5% bovine serum albumin for 30 min at room temperature before overnight incubation with the primary antibody at 4 °C. Fluorescence-conjugated secondary antibody was then incubated for 1 h at room temperature in the dark. The primary antibodies included CD11b antibody (Thermo Fisher Scientific Cat# 14–0112-82, RRID:AB_467108), Ly-6G/Ly-6C antibody (Thermo Fisher Scientific Cat# 14–5931-82, RRID:AB_467730), Cytokeratin 19 antibody (Proteintech Cat# 10,712–1-AP, RRID:AB_2133325). The nuclei were visualized using DAPI (C1006, Beyotime Biotechnology, Shanghai, China). The confocal microscope (LSM710, Zeiss, Jena, Germany) was used to obtain fluorescence images.

Pyrogallol (purity > 99.98%; HY‐N1579) (Figure 1A) and ML385 (HY‐100523) were obtained from MedChemExpress. The HO‐1 inhibitor ZnPP was purchased from AdooQ BioScience (Nanjing, China). MTT was purchased from Sigma‐Aldrich. DAPI (C1006) was obtained from Beyotime Biotechnology, Inc. (Shanghai, China). Recombinant human IFN‐β (#300‐02BC) was produced by Peprotech, Inc. (Rocky Hill, NJ, USA). Bead‐based multianalyte profiling kits for quantification of MIP‐1α, TNF‐α, MCP‐1, IL‐8, IP‐10, TRAIL, IL‐6, and RANTES were procured from Bio‐Rad Laboratories Inc. (Hercules, CA, USA). Antibodies are shown in Table S1.