Western blot analysis was performed using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis system. Protein samples (20 µg) were resuspended in reduced sample buffer, electrophoresed on a 7.5-10% Tris gel with Tris running buffer, blotted onto PVDF membranes, and sequentially probed with primary antibodies against RUNX2 (Cat. no. 12556; 1:1,000; Cell Signaling Technology), OCN (Cat. no. sc-365797; 1:1,000; Santa Cruz Biotechnology, Inc.), eukaryotic translation initiation factor 4E-binding protein 1 (4E/BP1; Thr37/46; Cat. no. 9644; 1:1,000; Cell Signaling Technology), phospho-S6 ribosomal protein (P-S6; S235/S236; Cat. no. sc-293143; 1:1,000), PPARγ (Cat. no. sc-7273; 1:1,000) (both from Santa Cruz Biotechnology, Inc.), C/EBPβ (Cat. no. 3802; 1:2,000), Sca-1 (Cat. no. 9664; 1:4,000), CD29 (Cat. no. 4706; 1:2,000), CD45 (Cat. no. 13917; 1:6,000), CD11b (Cat. no. 14271; 1:3,000) and β-actin (Cat. no. 3700; 1:2,500) (all from Cell Signaling Technology). Horseradish peroxidase-conjugated goat anti-rabbit (Cat. no. SAB4600223; 1:1,000) or anti-mouse (Cat. no. SAB4600004; 1:1,000) antibodies (both from Sigma) were added, and secondary antibodies were detected using enhanced chemiluminescence (ECL Plus; General Electric Healthcare, Milwaukee, WI, USA).
Cd11b
Manufactured by Cell Signaling Technology
Sourced in United States
CD11b is a cell surface glycoprotein that serves as an integrin alpha subunit. It plays a role in cell-cell and cell-extracellular matrix interactions.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (3 mentions)
Cd206, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
F4 80, by Cell Signaling Technology (2 mentions)
C ebpβ, by Santa Cruz Biotechnology (1 mentions)
Sca 1, by Cell Signaling Technology (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Runx2, by Cell Signaling Technology (1 mentions)
Dapi nuclear staining, by Merck Group (1 mentions)
Evos fl color imaging system, by Thermo Fisher Scientific (1 mentions)
Iba 1, by Cell Signaling Technology (1 mentions)
Slideview vs200, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
One step tunel apoptosis assay kit, by Meilun (1 mentions)
Tcs sp5 confocal scanning system, by Leica (1 mentions)
Casp1, by Santa Cruz Biotechnology (1 mentions)
Protein g magnetic beads, by Merck Group (1 mentions)
Cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Ne per nuclear, by Thermo Fisher Scientific (1 mentions)
15 protocols using cd11b
1
Comprehensive Western Blot Analysis
2
Microglia Phenotypic Characterization in Tissue
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Mice were sacrificed and perfused with 50 mL cold PBS, followed by 50 mL 4% paraformaldehyde. Cryosections were obtained of the coronal plain at 5 μm thickness. Non-specific staining was blocked by 1% BSA for 1 h, followed by incubation with primary antibodies against microglia surface marker CD11b (1:100 Cell Signaling, Danvers, MA, USA), M2 marker CD163 (1:120 Cell Signaling) for 2 h at 4 °C. Slides were washed thrice before staining with secondary antibodies: anti-rabbit (Cell Signaling) and anti-mouse (Cell Signaling). DAPI nuclear staining (Sigma-Aldrich) was added to the mounting solution. Primary-cultured microglia were stained as follows: cells were fixed with 4% paraformaldehyde for 1 h, washed thrice with cold PBS, and stained with antibodies against Iba-1(1:100, Cell Signaling) for 2 h with nuclear staining DAPI (Sigma-Aldrich) added at the last 15 min. Cells were washed twice before incubation with secondary antibodies: anti-mouse (Cell Signaling) and anti-rabbit (Cell Signaling). All images were taken by the Invitrogen EVOS FL Color imaging system.
3
Recombinant Protein Expression and Antibody Purification
4
Quantifying Immune Cells in Pancreatic Tissue
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Pancreatic tissues were stained with standard H&E preparations. Immunohistochemical analysis of paraffin-embedded pancreatic tissue was performed with an antibody specific for CD11b (Cell Signaling Technology) and CD8a (Proteintech). Immunostained tissues were photographed using a SLIDEVIEW VS200 (Olympus), and the average number of stained cells per field (total 3 fields, 2.6mm2 per field) was quantified.
5
Immunofluorescence Analysis of Neuroinflammation
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Brain sections were fixed in 4% paraformaldehyde (PFA) for 10 min, and the cell membrane was disrupted with 0.3% Triton X-100 for 10 min. Blocking was performed with 1% bovine serum albumin (BSA) for 1 h. Next, brain sections were incubated with antibodies against cluster of differentiation 11b (CD11b; 1:200, Cell Signaling Technology; Boston, MA, USA), CASP1 (1:200, Santa Cruz Biotechnology; Paso Robles, CA, USA), or ASC (1:200, Cell Signaling Technology) at 4 °C for 16 h. The sections were washed thrice with PBS and incubated at room temperature for 1 h with a secondary antibody corresponding to the primary antibody. The secondary antibody was washed off with PBS and stored at −20 °C after adding an antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies; Mulgrave, Australia). Deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed with a one-step TUNEL apoptosis assay kit (Meilunbio; Shanghai, China). Stained sections were observed with the TCS SP5 Confocal Scanning System (Leica; Wetzlar, Germany). Four fields along the peri-infarct regions were selected for each section.
6
Protein Extraction and Western Blotting
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Proteins were extracted from cell lysates using RIPA isolation buffer (Thermo Fisher Scientific, Inc). 30 µg of protein was loaded on a 4–15% gradient acrylamide gel, and gel electrophoresis and Western blots were performed using standard techniques26 (link). Blots were probed with CD3, CD11b, and CD11c antibodies (Cell Signaling Technologies, Inc. Danvers, MA).
7
Antibody Characterization for Flow Cytometry
8
Multiplex IHC Analysis of Tumor Microenvironment
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FFPE tumor tissue blocks were sliced into 4 mm-thick sections and placed on adhesion microscope slides (Matsunami). The tissue slides were deparaffinized and rehydrated prior to mIHC staining. Antigen retrieval and staining were performed using Opal 7-Color IHC Kits (AKOYA Biosciences) according to the manufacturer’s protocol. CD3 (Clone SP7; Abcam), CD8 (Clone C8/144B; Dako), CD206 (Clone CL0387; Invitrogen), CD11b (Clone D6×1N; Cell Signaling Technology), FOXP3 (Clone 236A/E7; Abcam), PDGFRα (Clone D1E1E; Cell Signaling Technology) and Cytokeratin (Clone AE1/AE3; Abcam) staining was examined and images were acquired using the Vectra 3 System (PerkinElmer). Images were exported using inForm Tissue Analysis Software (AKOYA Biosciences). Cell density and expression percentage of specific protein were calculated based on the average of at least three regions of interest (682 μm×510 µm/region) using HALO image analysis software (Indica Labs).
For PD-L1 staining using the anti-PD-L1 28–8 antibody, the combined positive score (CPS) was assessed by a pathologist (TKu) and defined as the percentage of total tumor cells (including tumor cells, lymphocytes, and macrophages) multiplied by 100 in the REGONIVO trial. In the TASNIVO trial, CPS was measured by the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies).
For PD-L1 staining using the anti-PD-L1 28–8 antibody, the combined positive score (CPS) was assessed by a pathologist (TKu) and defined as the percentage of total tumor cells (including tumor cells, lymphocytes, and macrophages) multiplied by 100 in the REGONIVO trial. In the TASNIVO trial, CPS was measured by the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies).
9
M2 Macrophage Differentiation and Co-culture
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THP-1 monocytes were differentiated into M2 macrophages by 12 h incubation with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich Chemical Company, St Louis MO, USA, P8139) followed by incubation with 20 ng/ml of interleukin 4 (IL-4) and 20 ng/ml of interleukin 13 (IL-13). M2 macrophage marker CD206 (#91992; rabbit; Cell Signaling Technology, Danvers, USA) and CD11b (#49420; rabbit; Cell Signaling Technology) were detected by flow cytometry to make sure M2 macrophage have been successfully polarized.
In co-culture experiments, 1 × 105 ovarian cancer cells were seeded in 6 Transwell chambers (#3450; Corning, NY, USA), and then put the inserts into 6-well plates containing differentiated THP-1 cells. The chamber was placed in a differentiated 6-well plate containing THP-1 cells and co-cultured for 48 h.
In co-culture experiments, 1 × 105 ovarian cancer cells were seeded in 6 Transwell chambers (#3450; Corning, NY, USA), and then put the inserts into 6-well plates containing differentiated THP-1 cells. The chamber was placed in a differentiated 6-well plate containing THP-1 cells and co-cultured for 48 h.
10
Aβ1-40 Regulation of Neuroinflammation
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The amyloid β protein fragment 1–40 (Aβ1−40), GW4869, PKH67, LPS and IFN-γ were purchased from Sigma (California, USA). Lipofectamine 2000 were purchased from Invitrogen (CA, USA). The antibodies GAPDH, YB-1, PTEN, NeuN and Iba1 were purchased from Abcam (Cambridge, MA), and the antibodies β3 tubulin, CD206, CD11b, Arg-1, iNOS were purchased from Cell Signaling Technology (Boston, Massachusetts). The ELISA kits were purchased from Blue gene (Shanghai, China). The YB-1 plasmids, miR-223 antagomir and miR-223 agomir were purchased from Genechem (Shanghai, China). The RiboCluster Profiler RIP-Assay Kit was purchased from MBL Medical & Biological Laboratories Co (LTD, Japan).
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