Mouse anti iba 1

The sections were washed with PBS to remove the cryoprotective solution. Subsequently, they were immersed in H₂O₂ to 0.33% in PBS for 30 min to block the activity of endogenous peroxidase. Sections were placed in a blocking solution (PBS with 0.5% bovine fetal serum, 0.3% Triton X-100 and 1% BSA) for 1 h and incubated overnight at 4°C with the corresponding primary antibody diluted in the blocking solution: mouse anti-β-galactosidase (1/5,000, Promega); mouse anti-PHF1 (1/200, anti-tau protein phosphorylated in ser396/404, 1/100, Peter Davis (Greenberg and Davies, 1990 (link)); mouse anti-caspase-3 (1/100, Invitrogen); mouse anti-Iba-1 (1/500, Wako); goat anti-Doublecortin (DCX; 1/500, Santa Cruz); or anti-calretinin (1/200, Swant).
The next day, the sections were washed three times for 10 min with PBS. They were then incubated first with the biotinylated secondary antibody and then with the avidin-catalase complex using the Elite Vectastain kit (SIGMAFASTTM DAB, Sigma, D4293). The developing reaction was performed using diaminobenzidine for approximately 10 min. Finally, the sections were placed in slides using FluorSave (Calbiochem, Merck Millipore) as a mounting medium. Images were taken using an Olympus BX41 transmitted light microscope that uses an Olympus ColorView IIIu CCD docked camera.

Paraffin‐embedded human OFC sections were deparaffinized, washed in PBS, and antigen retrieval performed by incubation in Citra solution (BioGenex) for 1 h at 70°C. Following incubation in blocking solution (MP Biomedicals), slides were incubated for 24 h at 4°C in a primary antibody solution consisting of blocking solution or Dako antibody diluent (Dako, Denmark) in combination with either rabbit anti‐TLR9 (1:70), rabbit anti‐pNF‐κB p65 (phospho S536) (1:150), mouse anti‐MCP‐1 (1:100), or mouse anti‐IL‐8 (1:300) combined with an antibody against neurons (chicken anti‐NeuN; 1:100; Novus; Cat. #NBP2‐10491), astrocytes (mouse anti‐GFAP; 1:50; Abcam; Cat. #ab4648 or rabbit anti‐GFAP; 1:250; Dako, Denmark; Cat. #Z0334), or microglia (mouse anti‐Iba‐1; 1:250; Wako, Osaka, Japan; Cat. #019‐19741). Slides were washed in PBS and incubated for 1 h at room temperature with the appropriate secondary antibodies (1:1000). Immunoblotting was visualized using Alexa Fluor 488 or 594 dye. Secondary‐only negative controls were performed without primary antibody incubation. Slides were coverslipped using Prolong Gold Anti‐fade mounting media (Life Technologies). Immunofluorescent images were obtained using a Nikon DS‐Ri2 scope (Nikon Inc.) and colocalization quantified using NIS Elements AR46 (Nikon Inc.).

Rats were deeply anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA) at desired time points post-eFSE. Brains were removed and post-fixed in 4% PFA for 90 min. Brains were then cryoprotected in 30% sucrose, rapidly frozen, and stored at −80°C. Thirty micrometer sections of dorsal hippocampus were obtained on a cryostat and stored in antifreeze at 4°C until use. Serial sections were blocked in 10% normal goat serum and 0.03% Triton X in 1× PBS for 1 h at 4°C. Primary antibodies were incubated in 4% normal goat serum with 0.03% Triton X overnight at 4°C. The following antibodies were used: rabbit anti-HMGB1 1:1000 (Abcam), mouse anti-GFAP 1:3000 (Millipore), and mouse anti-IBA1 1:4000 (Wako). Sections were washed with 1× PBS, and the reaction product was visualized using 3,3'-diaminobezidine. Colocalization of cell markers with HMGB1 was achieved by coincubating rabbit anti-HMGB1 1:1000 (Abcam) with the following antibodies: mouse anti-NeuN (Chemicon), mouse anti-GFAP 1:3000 (Millipore), and mouse anti-CD11b (ABD Serotec). After 24 h of incubation, sections were washed in 1× PBS and then incubated in the appropriate secondary antibodies conjugated with Alexa Fluor 568 or Alexa Fluor 488. Colocalization was visualized using confocal microscopy.

Spinal cords of at least 5 animals per genotype were dissected and lysed in RIPA buffer (50 mM Tris HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1% Triton X-100, 0.25% Na-Deoxycholate, 0.1% SDS, protease inhibitor cocktail). After 2 × 10″ sonication cycles, samples were incubated on ice and then centrifuged at 18000 × g for 20′ at 4 °C. Supernatants were then quantified with Bradford protein assay (Bio-rad) and resuspended in Laemmli Buffer before SDS-PAGE (Sigma-Aldricht). Antibodies used: rabbit anti-FUS (Bethyl), mouse anti-GFAP (Cell Signaling), mouse anti-Iba1 (Wako), rabbit anti-Gemin2 (Proteintech), mouse anti-Sm B/B’ (Thermo Scientific), mouse anti-SMN (BD), mouse anti-β-actin (Sigma).Anti‐rabbit and anti‐mouse IgG peroxidase‐conjugated secondary antibodies were from Bio Rad.

Cerebral cortex and hippocampus extracts were loaded onto 10-16 % Tris/tricine SDS gels, and transferred to nitrocellulose membranes before overnight incubation with one of the following primary antibodies: rabbit anti-APP (1:1000; Millipore), rabbit anti-Aβ_1–42 (1:1000; Sigma-Aldrich, Saint Louis, MO, USA) and mouse anti-SAPPβ (1:1000; Sigma-Aldrich), mouse anti-CTFβ (1:1500; Millipore), mouse anti-BACE1 (1:2000; Millipore), rabbit anti-PS1 (1:1000; Sigma-Aldrich), rabbit anti-NEP (1:1000; Millipore), rabbit anti-IDE (1:1000; Abcam, Cambridge, MA, USA), mouse anti-GFAP (1:2000; Millipore), mouse anti-Iba-1 (1:500; Wako), mouse anti-SYP (1:1500; Millipore), rabbit anti-PSD-95 (1:1000; Abcam), mouse anti-BDNF (1:500; Sigma-Aldrich) or rabbit anti-β-tubulin (1:3000; Sigma-Aldrich). Horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories) were used, and bands were visualized using ECL plus detection system. β-tubulin was utilized as an internal control for protein loading and transfer efficiency.