Summary of the primary antibodies used in the present study
| Antibody | Specificity | Immunogen/clone | Dilution | Source | Catalog/lot # |
|---|---|---|---|---|---|
| Flow cytometry | |||||
| 7/4 (recently identified as Ly-6B), PE-conjugated | 25–30 kDa GPI-anchored protein expressed in neutrophils and proinflammatory M1 monocytes | Clone 7/4 | 1:100 | AbD Serotec | MCA771PE |
| CD11b (Integrin αM chain, Mac-1 α chain), BD Horizon V450-conjugated | Murine αM chain of Mac-1 found on myeloid cells | Clone M1/70 | 1:333 | BD Biosciences | 560455/65504 |
| CD45 (Leukocyte Common Antigen, Ly-5), PerCP-conjugated | CD45 molecule on all leukocytes | Clone 30-F11 | 1:167 | BD Biosciences | 557235/51129 |
| Ly-6C, FITC-conjugated | 14–17 kDa GPI-anchored protein expressed at various levels in monocyte/macrophage subsets, granulocytes, and T cell subsets | Clone AL-21 | 1:500 | BD Biosciences | 553104/41637 |
| Ly-6G, PE-Cy7-conjugated | 21–25 kDa GPI-anchored protein expressed in neutrophils | Ly-6G-transfected EL4J cell line Clone 1A8 | 1:500 | BD Biosciences | 560601/44917 |
| Immunofluorescence labeling | |||||
| 7/4 | 25–30 kDa GPI-anchored protein expressed in neutrophils and proinflammatory M1 monocytes | Clone 7/4 | 1:800 | AbD Serotec | MCA771GA |
| Ionized calcium-binding adaptor molecule 1 (iba1) | 17 kDa EF hand protein expressed in macrophages and microglia | Synthetic peptide of C-terminus iba1 N′-PTGPPAKKAISELP-C′ | 1:750 | Wako | 019-19741/CDF1873 |
| Ly-6G | 21–25 kDa GPI-anchored protein expressed in neutrophils | Ly-6G-transfected EL4J cell line Clone 1A8 | 1:5000 | BD Biosciences | 551459/26029 |
| Immunoblotting | |||||
| β-Actin | Antibody reacts with all vertebrate actin isoforms | Purified chicken gizzard actin Clone C4 | 1:7000 | Millipore (Chemicon) | MAB1501 |
| Caspase-1 | Antibody reacts with full-length caspase-1 and cleaved forms that retain aa 371–390 | Synthetic peptide of amino acids 371–390 of caspase-1 (human) Clone 14F468 | 1:300 | Imgenex | IMG-5028/AB093004A-3 |
| Interleukin-1 α (IL-1α) | Based on amino acid composition, the theoretical MW of the recognized protein is 31 kDa | Highly pure (>98%) recombinant mouse IL-1α | 1:1250 | Abcam | ab9724 |
| Interleukin-1 β (IL-1β) | 17 kDa processed form of IL-1β | Synthetic peptide of N-terminal region of mature IL-1β (human) | 1:1000 | Cell Signaling | 2021 |
| Tumor necrosis factor α (TNF) | 26 kDa membrane-bound TNF precursor (pro-TNF) | Recombinant full-length TNF protein (human) | 1:1250 | Abcam | ab6671 |
| TNF-α converting enzyme (TACE) | Antibody reacts with mature, precursor and glycosylated forms of TACE (also termed Adam17) | Synthetic peptide of C-terminus TACE (human) | 1:300 | Lab Vision (NeoMarkers) | RB-1660-P/1660P807A |