C1005

Lung tissues were fixed in 4% paraformaldehyde overnight at room temperature, followed by paraffin embedding, sectioning (4 μm), deparaffinization, and rehydration. Immunodetection of neutrophils were performed on sections with the primary antibody goat anti‐human/mouse myeloperoxidase antigen affinity‐purified polyclonal antibody (RD system, AF3667) at 15 μg/ml for 3 h at room temperature. Then, cells were stained using NorthernLights 557‐conjugated anti‐goat immunoglobulin G secondary antibody (red, RD system, NL001) and counterstained with DAPI (blue, Beyotime, C1005).

12 mm diameter coverglass (Fisherbrand) was sterilized and pretreated with 1% gelatin (Sangon, A609764) in a 24-well culture plate for 30 min, and then prepared for seeding the cells. After the cells grew to 80% confluence, it was fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min and rinsed with PBS for three times. Then the cells were permeabilized with 0.25% Triton X-100 in PBS for 10 min and followed by blocking in PBST solution (1% BSA and 0.05% Tween-20 in PBS) for one h at room temperature. The primary antibodies were diluted in PBST solution and incubated overnight at 4 °C. Respectively, the appropriate secondary antibodies were diluted (in PBST) and applied for one h at room temperature. Each step was completed with 4 times PBS washing. DAPI (Beyotime, C1005) was used to present the nucleus and treated for 5 min. The cells were final mounted with anti-fade mounting medium (Beyotime, P0126) and detected immediately using a confocal laser scanning microscope (Carl Zeiss, CLSM 710). The negative control samples were treated without primary antibodies.

The mouse brain sections (6 μm) were incubated with primary antibodies against NOX2 (1:200, ab133303, Abcam), DHFR (1:200, ab124814, Abcam), GFAP (1:200, 2389127, Invitrogen, Carlsbad, CA, USA), and CD31 (1:200, ab24590, Abcam) at 4°C overnight. GFAP is considered a standard marker of mature astrocytes and it is often used to label astrocytes in IF. CD31 is also commonly used to label endothelial cells in IF. So, we used CD31 as a marker to distinguish endothelial cells and GFAP as a marker to distinguish astrocytes. The brain slices were incubated using the corresponding goat anti-rabbit IgG antibodies (1:250, SA00009-2, Proteintech) or goat anti-mouse IgG (1:250, SA00013-1, Proteintech) for 1.5 h at room temperature. Subsequently, the brain sections were sealed with anti-fluorescence solution after incubated DAPI (C1005, Beyotime) for 6 min. Finally, the sections were observed and captured under the fluorescence microscope. Immunofluorescence staining of hCMEC/D3 and HA was similar to brain sections after fixing cells with 4% paraformaldehyde.

For each treatment, a 300 µL cell suspension (10⁵ cells/mL) was seeded in an Eight-well Glass Chamber Slide (#80806, Ibidi, Grafelfing, Germany). Bufalin (40 nM) was added the next day and the medium was discarded after a specific time point (24 h for HCT116 and DLD1, and 48 h for SW480 and HCT15). The slides were then washed with cold PBS 3 times and fixed with neutral-buffered formalin for 10 min. The cell membrane was then permeabilized with 0.1% Triton-X (#KGF011, Keygen, Nanjing, Jiangsu, CN) for 10 min, washed with cold PBS 3 times and incubated with β-catenin primary antibody (aAb32672, Abcam, Cambridge, UK) diluted in 10% donkey serum (#SL050, Solarbio, Beijing, CN) overnight at 4 °C. The following day, the primary antibody was removed and the cells were washed with cold PBS 3 times. FITC-conjugated secondary antibody (donkey anti-rabbit IgG; #ab150073, Abcam, Cambridge, UK) diluted in 10% donkey serum was then added and incubated in darkness for 1 h. Next, the wells were washed 3 times with cold PBS and DAPI counterstaining was performed according to manufacturer instructions (#C1005, Beyotime, Shanghai, CN). Photos were taken at a magnification of 40x with a Leica DMI4000 B fluorescent microscope (Wetzlar, Germany). DAPI and FITC images were merged using ImageJ.

PKH67 dye was added to macrophage-EV/siHMGB1 according to the kit instructions (PKH67GL, Sigma Aldrich, St. Louis, MO, USA), which was incubated at room temperature for 15 min, and then centrifuged at 1000g for 5 min, followed by removal of the supernatant. The mixture was suspended in a macrophage-EV medium and centrifuged at 1000 for 5 min. Precipitate was obtained after repeating twice, which was PKH67-labeled macrophage-EV. Then, RAW264.7 cells were incubated in the dish pre-coated with the specific cell slides. When cell confluence reached 50%, PKH67-labeled macrophage-EV/siHMGB1 was added for incubation at 37 ℃ for 24 h. The cell slides were removed, washed three times in PBS, soaked with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 2% Triton X-100 for 15 min, and then stained by 4′,6-diamidino-2-phenylindole (DAPI; 2 μg/mL, C1005, Beyotime) for 10 min. Fluorescence expression was observed by confocal microscopy.
With the similar method mentioned above, Cy3-HMGB1 was loaded to the macrophage-EV. Macrophage-EV/Cy3-siHMGB1 was then co-cultured with RAW264.7 cells for 1 h and fixed in 4% paraformaldehyde. The nuclei were stained with DAPI. Internalization of macrophage-EV by RAW264.7 cells was observed under confocal microscopy.

After dewaxing and rehydration, 5 μm of paraffin sections were incubated with endogenous peroxidase at room temperature for 20 min, followed by antigen repair. Then the slices were incubated with 5% normal goat serum for 30 min, and went on incubating with diluted primary antibodies in 4 ℃ overnight. Next, the second antibodies were added to the slices, and cultured continuously at room temperature for 1 h. DAPI solution (C1005, Beyotime) was used to stained the cell nucleus for 10 min. Finally, the sections were sealed by resinene. The pictures of tissues were photoed using confocal microscopy (LSM800, Zeiss, German). Image J 1.49p was used to analyze the mean fluorescence intensity of TIPE2 and TAK1 in spinal cord and DRG. The detailed information of antibodies were as followed: (1) primary antibodies: rabbit anti-rat TIPE2 polyclonal antibody (1:550, 15940-1-AP, Proteintech), rabbit anti-rat TAK1 polyclonal antibody (1:80, bs-3585R, Bioss), mouse anti-rat TAK1 antibody (1:250, 67707-1, proteintech), rabbit anti-rat p-JNK1 + JNK2 (phospho T183 + Y185) (1:150, ab131499, Abcam), mouse anti-rat GFAP monoclonal antibody (1:600, 60190-1-lg, Proteintech); (2) second antibodies: goat anti-rabbit Cy3-labeled IgG (H + L) (1:500, A0516, Beyotime), goat anti-mouse FITC-labeled IgG (H + L) (1:500, A0568, Beyotime).

In fixed cell cultures, 0.3% Triton X-100 (ST795, Beyotime Institute of Biotechnology, China) was added and incubated at 37°C for 5 min. Goat serum (C0265, Beyotime Institute of Biotechnology, China) was then added and incubated at room temperature for 60 min. Subsequently, the sections were incubated with antibodies against β-catenin (A19657, ABclonal Technology, China), CHMP4C (bs-7744R, Bioss Biotechnology Co., Ltd., China), GSK3β (A2081, ABclonal Technology, China), and p-GSK3β (bs-3161R, Bioss Biotechnology Co., Ltd., China) overnight at 4°C. After washing with Phosphate-Buffered Saline (PBS) (G0002, Sevier Biotechnology Co., Ltd., China), FITC Goat Anti-Rabbit IgG (H + L) (AS024, ABclonal Technology, China) was applied and incubated in the dark at 25°C for 1.5 h. Sections were subjected to 4′,6-diamidino-2-phenylindole (DAPI) staining (C1005, Beyotime Institute of Biotechnology, China), followed by a 5-min incubation in the dark and removal of excess DAPI through PBS wash. Subsequently, these sections were sealed with an anti-fade mounting medium (P0126; Beyotime Institute of Biotechnology, China). Ultimately, an inverted fluorescence microscope (ICX41; Sunny Optical Technology (Group) Co., Ltd., China) was employed to observe and capture images of the sections.