Phospho nf κb ser536

Protein extracts were obtained using RIPA buffer supplemented with protease (Sigma‐Aldrich) and phosphatase (Thermo Fisher Scientific) inhibitor cocktails. Western blot analyses were performed using antibody against total NF‐κB, phospho‐NF‐κB at serine 536 (phospho‐NF‐κB^Ser536; Cell Signaling Technology), Caspase‐1 (Santa Cruz Biotechnology) and lamin B1 (nuclear loading control; Abcam), as previously described.¹³

Thirty-micrometer-thick sagittal cryosections of mouse brain were cut and mounted on glass slides. The sections were pretreated with 0.01 mol/L citrate buffer (pH = 6.0) for 20 min and then were blocked for 1 h with 10% goat serum in PBST (0.25% triton-X100). Following blocking, the sections were incubated with primary antibodies, overnight at 4 °C. The next day, the sections were washed and incubated with Alexa-Fluor fluorescent dye-conjugated secondary antibodies Invitrogen, USA; 1:500) in PBS for 90 min at room temperature. The primary antibodies were specific to mouse monoclonal human TDP-43 (Abnova, Taiwan; 1:500), mouse polyclonal GFAP (Cell Signaling Technologies, USA; 1:500), rabbit polyclonal Iba1 (Wako Chemicals, USA; 1:500), mouse monoclonal NeuN (Cell Signaling Technologies, USA; 1:1000) Nuclear factor kappa-B (NF-κB) (Santa Cruz;1:1000), phospho-NF-κB (Ser 536) (Cell Signaling Technologies, USA; 1:500), arginase-1 (Santa Cruz; 1:1000), and Ym-1 (Stem Cell Technologies, Canada;1:500) protein markers. Four equidistant sections for the hippocampus and six equidistant sections of the cortex were assessed for every mouse. All sections were imaged using confocal microscopy (LSM microscope 700, Zeiss). The ImageJ software was used for analyzing and quantifying the immunoreactive areas.