Mouse anti cd11b

Necrostatin-1 (Nec-1) was purchased from Sigma (M6006). The other chemical inhibitors including the fluorescent-labelled ligand 3-(3-((3-(4-amino-5-(4-(3-(2-fluoro-5-(trifluoromethyl)phenyl)ureido)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)propyl)amino)-3-oxopropyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide were synthesised by Takeda Chemical Industries, Ltd²² . Z-VAD-FMLK at a 20 mM stock solution in neat dimethyl sulfoxide (DMSO) was purchased from Promega (G7232) and Smac mimetic (AT-406) was from AdooQ BioScience (A11163-10). Recombinant mouse and rat TNF-α were purchased from Wako and R&D systems (410-MT-050 and 510-RT-050, respectively). The following antibodies were used for ELISA: mouse MLKL (MABC604, Millipore), mouse phospho-MLKL/S345 (ab196436, Abcam), and rabbit IgG HRP (711-035-152, Jackson ImmunoResearch). TMB was purchased from KPL (53-00-0302). The following antibodies were used for fluorescence-activated cell sorting (FACS) experiments: mouse anti-CD11b (553310, BD Biosciences; 1:200 dilution), and Ly-6G (127608, Biolegend; 1:400).

The brain sections were incubated in blocking solution for 2 h at room temperature, and then incubated at 4 °C overnight with one of the following antibodies: mouse anti-CD31 (1:100; BD Pharmingen), rabbit anti-ZO-1 (1:100; Invitrogen), rabbit anti-occludin (1:100; Invitrogen), rabbit anti-claudin-5 (1:50; Invitrogen), mouse anti-glial fibrillary acidic protein (GFAP, 1:1000; Millipore), mouse anti-CD11b (1:100; BD Pharmingen), rabbit anti-VCAM-1 (1:100; Santa Cruz), and rabbit anti-ICAM-1 (1:100; Santa Cruz). After five washes in 0.1% Triton X-100 in PBS for 15 min each, the sections were incubated with secondary antibody overnight at 4 °C. Before washing, the sections were treated with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) and washed five more times with 0.1% Triton X-100 in PBS for 30 min each. All antibodies were dissolved in antibody diluent (Dako). Confocal images were captured at room temperature with ZEN software on an upright confocal microscope (LSM 700; Carl Zeiss) using the predefined ZEN software configurations for Alexa Fluor 546, Alexa Fluor 488, and DAPI.

A marker of microglial activation, an antibody to CD11b (OX-42), was used to identify areas of inflammation in the rat brain. Three groups of rats (n = 3/group) were used to assess the effects of JM6 on TBI-induced inflammation in the hippocampus using immunohistochemistry: 1) TBI; 2) SHAM; 3) TBI + JM6. Rats were prepared and treated as above. Twenty-four hours after injury, rats were perfused with 4% paraformaldehyde, brains collected and 10-μm frozen sections were cut on a cryostat. Sections were then incubated overnight with a primary antibody (mouse anti-CD11b; 1:2000, BD Biosciences, San Jose, CA). The following morning, sections were rinsed and then incubated with a secondary antibody (Alexa 594 goat anti-mouse; 1:400, Life Technologies, Grand Island, NY) at ambient temperature, and then mounted with DAPI (marker of nuclei) for imaging. An Olympus BX51 Fluorescent Microscope was used to visualize the hippocampal formation and surrounding cortical regions.