Anti il 1β

Cells were fixed with 4% paraformaldehyde for 10 min and then washed in phosphate-buffered saline (PBS). After being blocked with 5% bovine serum albumin containing 0.5% Triton X-100 for 1 h at 37°C, the microglia were incubated with the following primary antibodies at 4°C overnight: mouse anti-Iba1 (1:200, Bioss, Beijing, China), rabbit anti-iNOS, anti-Arg1, anti-IL-1β, anti-TNF-α, anti-IL-4, anti-IL-6 and anti-IL-10 (1:200, Bioss). After being washed in PBS three times, the cells were incubated with the following secondary antibodies at 37°C for 1 h: Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, Bioss) or Alexa Fluor Cy3-conjugated goat anti-mouse IgG (1:200, Bioss). After being washed in PBS three times, the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Biyuntian, China) for 5 min. The cells were then washed in PBS and observed under a 7266-fluorescence microscope (Leica, Japan).

Enzyme-linked immunosorbent assay (ELISA) was performed to determine the proteins found in or on the EVs before and after P. aeruginosa infection. EVs (40 μg) were bound in 100 µL bicarbonate buffer (pH 9.5) in 96-well plates and incubated at 4 °C overnight. The following day, each plate was washed three times in 200 µL washing buffer (0.05% Tween-20 in PBS). The plate was then blocked using 100 µL blocking solution (5% non-fat dry milk and 0.05% Tween-20 in 1× PBS) for 1 h. After blocking, the primary antibodies anti-HSP70 (Santa Cruz, catalog no. sc-32239), anti-HSP 90β Thermo Scientific, catalog no. 37-9400), anti-cleaved caspase 3 (R & D, catalog no. MAB835), anti-LAMP-1 (DSHB, catalog no. 1D4B), anti-IL-6 (DSHB), anti-IL-1β (Bioss antibodies, catalog no. bs-0812R-FR), and anti-TSG101 (Thermo Fisher, catalog no. MA1-23296) were added to each sample with blocking buffer and incubated at room temperature for 2 h. After incubation, each plate was washed, horseradish peroxidase secondary antibody (Dako) was added, and the plate was incubated at room temperature for 30 min. The ELISAs were developed using SigmaFast o-phenylenediamine dihydrochloride peroxidase substrate (Sigma Aldrich) and read at a 405-nm wavelength using a Genemate UniRead 800 microplate reader.

Sections were stained with the following primary antibodies for immunohistochemical analyses: anti-TNF-α (dilution ratio: 1:400; Bioss Inc., Woburn, MA, USA), anti-IL-1β (1:400; Bioss Inc.), anti-TGF-β1 (1:400; Bioss Inc.), anti-OPN (1:400; Abcam, Cambridge, UK), and anti-CTSK (1:400; Abcam). Briefly, after deparaffinization and rehydration, the sections were incubated with 3% hydrogen peroxide (Abcam) to quench the endogenous peroxidase activity; thereafter, the sections were blocked with specific animal serum for nonspecific binding and then incubated with specific primary antibodies and VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Laboratories, Inc., Burlingame, CA, USA) with biotinylated secondary antibody and ABC Reagent. Subsequently, 3,3′-diaminobenzidine (Abcam) was applied, and hematoxylin (Fujifilm Wako Pure Chemical Corp.) staining was performed for counterstaining. The levels of TNF-α, IL-1β, TGF-β1, and OPN were semi-quantified by the percentage of immuno-positive stained areas (%).

We used the following materials: Con A was acquired from Sigma-Aldrich (St. Louis, MO). BBR, alcian blue stain kit, methylprednisolone (MP), ampicillin, neomycin, metronidazole, and vancomycin were afforded by Solarbio (Beijing, China). Anti-Caspase-3, anti-Bax, and anti-F4/80 antibodies were offered by cell signaling technology (Boston, MA). Anti-TNF-α, anti-interferon-γ, anti-IL-1β, anti-IL-17A, and anti-IL-10 antibodies were purchased from Bioss (Beijing, China). Rabbit anti-mouse primary antibodies, including zonula occludens-1 (ZO-1), occludin, TLR4, NF-κB P65, inhibitor of NF-κB (IκB), phospho-inhibitor of NF-κB (p-IκB), and β-actin, were obtained from Abcam (Cambridge, UK). Allophycocyanin-conjugated anti-CD4, fluorescein isothiocyanate–conjugated anti-IL-17A, and phycoerythrin-conjugated anti-Foxp3 antibodies were purchased from BD (Franklin Lakes, NJ). LPS ELISA kits were supplied by Cloud-clone (Hubei, China).

Monoclonal Anti-Actin and Anti-NLRP3 antibodies were obtained from Sigma-Aldrich (St. Louis, MO). Mitosox Red, Mitotracker Red CMXRos, CMH₂-DCFDA, 10-N-nonyl acridine orange (NAO), LysoSensor Green DND-189, tetramethylrhodamine methyl ester (TMRM), CellTracker™ Green and Hoechst 33342 were from Invitrogen/Molecular Probes (Eugene, OR). Anti-GCase was obtained from Abcam. Anti-cytochrome c antibody was obtained from BD Biosciences Pharmingen (San Jose, CA) and anti-GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) monoclonal antibody (clone 6 C5) was from Calbiochem-Merck Chemicals Ltd. (Nottingham, UK). CBE, Anti-MAP LC3 (N-20), anti-LAMP-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Protease inhibitors were from Boehringer Mannheim (Indianapolis, IN). Anti-IL-1β were obtained (Bioss, Inc). Anti-Caspase 1 was obtained from (Cell Signaling Tecnology, CST). The anti-GlcCer rabbit anti-serum was purchased from Glycobiotech GmbH (Kükels, Germany). Glucocerebrosides from Gaucher’s spleen (GlcCer) was obtained from Matreya LCC (Pleasant Gap, PA, USA). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA, USA). All other chemicals were purchased from Sigma-Aldrich.

At different reperfusion times, protein was extracted from the infarcted hemispheres and cultured cells by a protein extraction kit (Beyotime Biotech) as described in our previous report (Li et al., 2015 (link)). Protein samples were next electrophoresed and transferred to polyvinylidene difluoride filter (PVDF) membranes. Then block the membrane with 5% fat-free milk at room temperature for 2 h, and incubate with primary antibodies as follows: anti-Tubulin (Cell Signaling), anti-Cx43 (Cell Signaling), anti-TLR4 (Proteintech), anti-MyD88 (Cell Signaling), anti-NF-κB p65 (Proteintech), anti-IL-1β (Bioss), and anti-TNF-α (Bioss) at 4°C overnight. Next day, incubate with secondary horse anti-mouse or goat anti-rabbit antibodies conjugated with horseradish peroxidase (Cell Signaling) for 2 h and visualize by an enhanced chemiluminescence system (ECL). Relative protein levels were quantified after normalization to Tubulin.