Ab60176

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).

For immunofluorescence, frozen sections were labeled with unconjugated primary antibodies against MOMA-2 (1:200, MCA519G; AbD) and a M1 marker [iNOS (1:200, ab178945; Abcam) or CD86 (1:200, NBP2-25208; Novus)], or a M2 marker [Arg1 (1:200, ab60176; Abcam) or CD206 (1:200, ab64693; Abcam)] simultaneously overnight, followed by incubation with a fluorophore-conjugated secondary antibody for 30 min. The stained sections were mounted with DAPI-containing VectorShield mounting medium (Vector) and then viewed using an Olympus BX53 fluorescence microscope.

Total protein was extracted from 20 μg of frozen tissue in 400 μl of protein lysis buffer by homogenization on ice. The homogenates were then transferred to pre-chilled 1.5-ml EP tubes, placed on ice for 15 min for complete lysis, and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatants were transferred to 0.5-ml centrifuge tubes and stored at −20°C. A 50- to 100-μg sample was mixed with 5 × loading buffer, heated in boiling water for 5 min, quickly cooled, and loaded onto a polyacrylamide stacking gel for electrophoresis. The stacking gel was run at 80 V and separation gel was run at 120 V. The separated proteins were then transferred to the polyvinylidene difluoride (PVDF) membranes. The membranes were then washed with tris-buffered saline and 1% Triton (TBS-T) for 5 min, followed by incubation with the primary antibody diluted in blocking solution at 4°C overnight. The membranes were washed three times with TBS-T (5 min per wash) and then incubated for 2–3 h with a horseradish peroxidase (HRP)-labeled secondary antibody (Abcam, Lot No. Ab60176; Abcam, Lot No. Ab15323) diluted in blocking buffer. The blotted membranes were washed again with TBS-T (three times, 5 min each), and the bands were visualized with ECL chemiluminescence reagent. The gray-scale images were analyzed using imaging software.

OECs and microglia grown on the coverslips were treated according to the experimental protocols. Then the immunofluorescence was performed following previously described methods (Wang et al. 2022 (link); Guo et al. 2020 (link)). The primary antibodies were used to stain cells, including anti-p75 (ab245134, 1:300, Abcam), Iba1 (019-19741, 1:500, Wako), iNOS (ab49999, 1:300, Abcam), Arg-1 (ab60176, 1:300, Abcam), TREM2 (ab95470, 1:500, Abcam), F4/80(ab6640, 1:200, Abcam), pNF-κB (3033, 1:200, Cell Signaling Technology), APOE (ab183597, 1:200, Abcam) and DAPI (ab228549, 1:1000, Abcam). After incubation with the corresponding secondary antibodies, coverslips were washed and mounted on the slides. All images were captured by fluorescence microscope (Leica Microsystems, Germany).