Ab153696

Brains were postfixed overnight in 4% PFA before being dehydrated with sucrose solution (10%, 20%, 30%) for 3 days at 4 ℃. Brains were cut into 50-μm-thick sections in the coronal plane. Sections containing DLS and DMS were rinsed with 0.5% Triton X-100 (Solarbio, China) for 5 min, and blocked with 5% BSA (Sangon Biotech, China) for 1 h at room temperature. Then, the sections were incubated with mouse anti-CD31 (ab64543, 1:200, Abcam, UK), rabbit anti-claudin 5 (ab15106, 1:200, Abcam, UK), chicken anti-GFAP (ab4674, 1:2000, Abcam, UK), rabbit anti-Iba1 (ab153696, 1:500, Abcam, UK), rabbit anti-IL17A (A0688, 1:50, Abclonal) and mouse anti-CD4 (67786-1-lg,1:200, Proteintech) antibodies for 48 h at 4 ℃ in the dark. Then, the sections were incubated with Alexa-568 donkey anti-rabbit IgG (ab175470, 1:1000, Abcam, UK), Alexa-488 donkey anti-mouse IgG (ab150105, 1:1000, Abcam, UK), and Alexa-594 goat anti-chicken IgG (ab150172, 1:1000, Abcam, UK) for 2 h at room temperature. Finally, sections were mounted on slides with Fluoroshield™ with DAPI (F6057, Sigma-Aldrich, USA). Images were captured with a Nikon A1R HD25 confocal microscope system (Nikon, Japan). Immunofluorescence quantification was performed as previously described [24 ] and detailed in the Additional file 1.

The immunofluorescence assay was conducted following the previously reported methods with minor changes (Wu et al., 2019 (link)). The paraffin-embedded slices were obtained in the same way as above. Similarly, the slices were permeated with 0.3% Triton X-100 for 30 min and blocked them with 5% BSA for 25 min, followed by the incubation with anti-Iba-1 (ab153696, 1:500, Abcam; sc-32725, 1:100, Santa Cruz Biotechnology), anti-CD86 (sc-28347, 1:100, Santa Cruz Biotechnology), anti-iNOS (ab178846, 1:500, Abcam), anti-CD206 (60143-1-1g, 1:100, Proteintech), anti-CD163 (ab182422, 100, Abcam) and anti- p-JNK (sc-6254, 1:200, Santa Cruz Biotechnology) overnight at 4°C. The slices were then incubated with the secondary antibodies for nearly 30 min at RT. Cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, MA, United States). The “sham”, “control” and “vehicle” were applied as the controls. The images were acquired by a fluorescence microscope (Olympus OX51).

After xylene deparaffinization, the hippocampus tissue sections were washed by 0.1M PBS and microwave repaired for 15 min, blocked with 10% normal goat serum for 20 min, followed by the addition of 300 µL of primary antibodies (rabbit anti-rat Iba-1; 1:200; ab153696, Abcam, Cambridge, UK) for overnight incubation. After washing by PBS, sections were incubated with a fluorescent secondary antibody (Alexa Fluor^® 488-labeled Goat anti rabbit; 1:300; ab150077, Abcam, Cambridge, UK) for 2 h in the dark, washed three times with PBS, and mounted using anti-fluorescence quencher containing DAPI. Ultimately, a confocal laser-scanning microscope was utilized to observe and photograph the sections (magnification, ×200; Olympus FV1000, Olympus, Tokyo, Japan).

The protein was extracted from brain tissues using an Ambion PARIS kit (Solarbio, Beijing, China) with addition of phosphatase and protease inhibitor cocktails (Roche). The Pierce BCA Protein Assay Kit (Cat.#23227, Thermo Fisher) was used to detect protein concentrations. The proteins were separated by SDS-PAGE electrophoresis, and a blotted membrane was incubated with blocking buffer and then with anti-Claudin 5 (1 : 500, ab15106, Abcam, USA), anti-Iba1 (1 : 1000, ab153696, Abcam, USA), anti-GFAP (1 : 1000, Cat.#12389, CST, USA), anti-MBP (1 : 1000, Cat.#13344, CST, USA), or anti-NF-L (1 : 1000, Cat.#AB9568, Millipore, USA) primary antibody at 4°C overnight before addition of horseradish peroxidase- (HRP-) linked goat anti-rabbit (1 : 1000, Cat.#7074, CST, USA) or goat anti-mouse secondary antibody (1 : 25000, Cat.E030110-01, EarthOx, USA). Detection of β-tubulin was performed on stripped membranes with anti-β-tubulin (1 : 1000, ab179513, Abcam, USA) primary antibody to control for protein loading. Protein signal was visualized with the LumiGLO chemiluminescent substrate (Cat.#7003S, CST, USA), and target protein expression was normalized as fold change relative to β-tubulin expression using Photoshop for quantitative analysis.

N-acetylcysteine (NAC; A7250), dimethyl sulfoxide, 4′,6-diamidino-2-phenylindole, and anti-β-actin (A1978) were from Sigma-Aldrich (St. Louis, MO). Recombinant human prorenin was from Abcam (ab93266; Cambridge, MA) and MCC950 (sc-505904) was from Santa Cruz Biotechnology (Santa Cruz, CA). The ROS fluorescent probe-DCFH-DA kit (S0033) and caspase 1 activity assay kit (C1101) were from the Beyotime Institute of Biotechnology (Nanjing, China). The following antibodies were used: sheep monoclonal antiserum against prorenin/renin (GTX7967, Gene Tex, San Antonio, TX); rabbit polyclonal antiserum against PRR (bs-7691R, Bioss, Beijing, China); rabbit polyclonal antiserum against caspase-1 (D7F10); rabbit anti-ASC monoclonal antibody (#13833), anti-CD86 (#91882), and anti-CD206 (#91992) were from Cell Signaling Technology (Beverly, MA); mouse monoclonal antiserum against CD11b/c (OX42, ab1211), mouse monoclonal to IL-6 (ab9324), iNOS (ab15323), Arg (ab60176), Iba-1 (ab153696), GFAP (ab7260), PGP 9.5 (ab10410), and rabbit polyclonal antiserum against NLRP3 (ab214185) were from Abcam. Donkey anti-sheep IgG H&L Alexa Fluor 555 (ab150178), goat anti-rabbit IgG H&L Alexa Fluor 594 (ab150080), and goat anti-mouse IgG H&L Alexa Fluor 488 (ab150117) were from Abcam. MCC950 was from Adipogen Corp. (San Diego, CA). PLX5622 was from Plexxikon (Berkeley, CA).

Brains were removed and fixed with 4% paraformaldehyde at 4 °C overnight. Frozen sectioning was performed after sucrose gradient dehydration followed by blocking with 10% goat serum containing 0.5% Triton X-100 at room temperature for 30 min. Brain sections were incubated with different primary antibodies at 4 °C overnight, including anti-MPO (Abcam, ab90812, 1: 1000), anti-NeuN (Abcam, ab177487, 1: 1000), anti-CD31 (Abcam, ab222783, 1: 1000), anti-Iba-1 (Abcam, ab153696, 1: 1000), anti-GFAP (Abcam, ab7260, 1: 1000) antibodies. The sections were washed with PBS 3 times and incubated with the mixture of Alexa Fluor 488 conjugated goat-anti-mouse IgG (Beyotime, A0428, 1: 500) or Alexa Fluor 555 conjugated donkey-anti-rabbit IgG (Beyotime, A0453, 1: 500) for 1 h in darkness at room temperature, followed by staining with DAPI for 5 min. After being washed with PBS 3 times, sections were mounted with fluorescent mounting medium (Dako, S3023), and examined by a confocal laser scanning microscope (LSM 780, Carl Zeiss).