Cocultures were completed and macrophages were fixed as described above. Coverslips were washed once with PBS prior to staining with SYTOX Green (final concentration, 10 μM) (ThermoFisher) for double-stranded DNA (dsDNA), and Hoechst 33342 (final concentration, 5 μM ) (ThermoFisher) for condensed chromatin (nuclei). Additional staining for histones and MMPs was accomplished by blocking cells in 1% bovine serum albumin in PBS for 30 min at 37°C followed by a 1-h incubation at 37°C with antibodies for histone H3 (ab5103; Abcam, Cambridge, MA), neutrophil elastase (ab68672; Abcam), myeloperoxidase (ab9535; Abcam), matrix metalloproteinase 1 (MMP-1) (ab551168; Abcam), MMP-7 (ab5706; Abcam), MMP-8 (ab81286; Abcam), MMP-9 (ab38898; Abcam), or MMP-12 (ab137444; Abcam). Cells were then washed three times with 1% BSA in PBS, followed by a 30-min incubation with an Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (ThermoFisher) and two additional washes with 1% BSA in PBS prior to mounting coverslips onto glass microscope slides with Aqua Poly/Mount (Polysciences Inc., Warrington, PA). Macrophages were visualized with a Zeiss LSM 710 META inverted laser scanning confocal microscope, and extracellular traps were identified by dsDNA staining that extended into the extracellular environment.
Ab81286
Manufactured by Abcam
Sourced in United Kingdom
Ab81286 is a recombinant monoclonal antibody that recognizes the human Vimentin protein. Vimentin is a type III intermediate filament protein that is expressed in various cell types, including mesenchymal cells, endothelial cells, and some hematopoietic cells. The antibody can be used for the detection of Vimentin in various applications, such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Sytox green, by Thermo Fisher Scientific (3 mentions)
Ab38898, by Abcam (2 mentions)
Axiocam hrc camera, by Zeiss (2 mentions)
Ab73734, by Abcam (2 mentions)
Axioimager mi microscope, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab9535, by Abcam (1 mentions)
Ab5706, by Abcam (1 mentions)
Ab137444, by Abcam (1 mentions)
Ab68672, by Abcam (1 mentions)
Ab5103, by Abcam (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lsm 710 meta inverted laser scanning confocal microscope, by Zeiss (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab58425, by Abcam (1 mentions)
Ab66157, by Abcam (1 mentions)
Ab182793, by Abcam (1 mentions)
Ab68185, by Abcam (1 mentions)
Ab186421, by Abcam (1 mentions)
9 protocols using ab81286
1
Macrophage Extracellular Trap Visualization
2
Immunohistochemical Analysis of Osteoclast Markers
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Osteoclasts cultured on dentine or cartilage were fixed in 4% formalin. Dentine discs were decalcified in 0.5 M EDTA prior to paraffin-embedding. H&E staining was performed on transverse 5 µm sections. Antigen retrieval of deparaffinised OA tissue sections was performed in hot citric acid solution. Sections were exposed to primary rabbit monoclonal antibodies against MMP-8 (ab81286, 1:1000, Abcam, Cambridge, UK) or MMP-9 (ab73734, 1:1000, Abcam) overnight at 4 °C and staining was visualised with 3,3′-diaminobenzidine (DAB). Image acquisition was performed using a Zeiss AxioImager MI microscope, AxioCam HRC camera and AxioVision software. Osteoclasts in tissue sections were considered as large, multinucleated cells containing ≥ 3 nuclei; a widely accepted identification criterion18 (link),37 (link),38 (link).
3
Immunohistochemical Analysis of Lung MMPs and TIMPs
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Expression of MMP-8, MMP-9, MMP-12, TIMP-1, and TIMP-4 in rat lung was assessed by immunohistochemistry. Briefly, paraffin embedded lung tissue slice was deparaffinized and rehydrated. Antigen was retrieved using sodium citrate and with heat-induced retrieval. After blocking with goat serum, antiMMPs or TIMPs antibodies at 1:200 dilution (all antibodies were purchased from Abcam, Shanghai, People’s Republic of China – antiMMP-8 antibody: ab81286; antiM-MP-9 antibody: ab76003; antiMMP-12 antibody: ab66157; antiTIMP-1 antibody: ab16412; antiTIMP-4 antibody: ab58425) were applied overnight at 4°C. After horseradish peroxidase-conjugated second antibody was applied, expression of MMPs and TIMPs was visualized using a DAB detection kit. Images of MMP-8, MMP-9, MMP-12, TIMP-1, and TIMP-4 were obtained and photographed under microscope (Olympus Corporation).
4
Western Blot Analysis of Inflammatory Markers
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The tissues were fully lysed and sonicated, and the total protein was extracted. Protein levels were quantified using a BCA Protein Assay Kit. Polyacrylamide gel electrophoresis was performed with 10 μg protein from each sample. Next, proteins on the gel were transferred onto a polyvinylidene fluoride (PVDF) membrane by semi-dry transfer, and the PVDF membrane was sealed with 5% skim milk powder for 2 h at RT. Thereafter, the membrane was probed with the primary antibody at 1:1000 dilution with phosphate buffer saline (PBS) against matrix metalloproteinase 8 (MMP8), MMP9, IL-17β, tissue inhibitor of metalloproteinase 1 (TIMP1), Ccl2, Ccl7, TNF ligand superfamily member 9 (TNFSF9), and Fas apoptotic inhibitory molecule (FAIM), S100a8, S100a9 at 4 °C overnight. All primary antibodies were provided by Cell Signaling. The PVDF membrane was then washed three times with PBS-Tween for 8 min each. The secondary antibody was diluted with PBS and was incubated for 1 h at RT, and the PVDF membrane was washed again. β-actin was used as a loading control. Proteins were visualized using an enhanced chemiluminescence solution. The following antibodies were used: MMP8 (Abcam, ab81286), MMP9 (CST, 13667T), IL-17β (Abcam, ab125029), TIMP1 (CST, 8946S), Ccl2 (Abcam, ab186421), Ccl7 (Abcam, ab182793), TNFSF9 (Abcam, ab68185), FAIM (Abcam, ab154570), S100a8 (CST, 47310T), and S100a9 (CST, 73425S).
5
Protein Expression Analysis of NETs
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A sample containing 80 μg of the extracted proteins from the NETs induced by PMA, S. suis or PBS was subjected to a Western blot analysis with antibodies against PGRP-S (sigma, SAB2500783), WDR5 (sigma, PLA0256), Lysozyme (abcam, ab158508), Calpain (abcam, ab108400), or MMP8 (abcam, ab81286). The expression of GAPDH was also determined as a reference control with a GAPDH antibody (Calbio, CB100127).
6
Western Blot Analysis of Extracellular Matrix Proteins
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We extracted the total protein by RIPA lysate. Then, we separated 30 μg protein on 10% protein electrophoresis gel and then transferred it to the PVDF membrane (Invitrogen). The PVDF membrane was blocked with 5% skim milk powder for 1-2 h, and then anti-collagen III (COLIII) (1 : 1000, Abcam, ab184993), anti-α smooth muscle actin (α-SMA) (1 : 500, Abcam, ab5694), anti- matrix metalloproteinase (MMP)-1 (1 : 1000, Abcam, ab52631), anti-MMP-8 (1 : 1000, Abcam, ab81286), anti-NFAT5 (1 : 1000, Abcam, ab3446), antitumor necrosis factor-α (TNF-α) (1 : 1000, Abcam, MA5-23720), interleukin (IL)-6 (1 : 500, Abcam, M620), anti-IL-1β (1 : 500, Abcam, M421B), and anti-β-actin (1 : 1000, Abcam, ab6276) were added and incubated overnight 4 °C. The next day, the PVDF membranes were incubated in HRP anti-mouse IgG for IP (1: 1000, Abcam, ab131368) at 25 °C for an h. The bands were detected using an exposure meter (Bio-Rad, USA) with hypersensitive chemiluminescence (HRP) detection kit (LMAI Bio, Shanghai, China).
7
Quantification of Pro and Active MMP-8
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Samples were separated on Criterion XT Precast 26-well 4–12% SDS-PAGE gradient gels at 30 V for 10 min and then at 180 V for 1 h, and electrotransferred at 100 V for 1 h at 4°C onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The nitrocellulose membrane was first stained with Ponceau S (0.1%, w/v) in 1% acetic acid for 6 min at ambient temperature, photographed, washed thoroughly in distilled water (dH2O), blocked with an Odyssey buffer, incubated for 18 h at 4°C with monoclonal rabbit antibody against human MMP-8 (ab81286; Abcam Cambridge, UK) diluted 1:1000, and then with goat antirabbit DyLight® (Thermo Fisher Scientific, Waltham, MA, USA) 800-conjugated secondary antibody diluted 1:5000 for 1 h at ambient temperature (22 (link)). The immunoreactions were visualized using infrared imaging and band intensities were estimated by densitometry (ImageJ, NIH, Bethesda, MD, USA). The amount of pro MMP-8 and active MMP-8 molecular species were estimated by comparison with the intensity of rhMMP-8 (908-MP, R&D Systems, Minneapolis, MN, USA). The proportion of active MMP-8 to total MMP-8 (sum of pro and active MMP-8) was calculated and expressed as a percentage. The Western blot analysis was repeated 3 times.
8
Liver Tissue Protein Expression Analysis
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Liver tissue was lysed in ice-cold lysis buffer (Beyotime, China) containing protease inhibitors (Beyotime, Shanghai, China). Total protein was measured using a bicinchoninic acid protein assay (Beyotime, China). Then, the lysates were loaded onto 10% SDS-polyacrylamide gels. After electrophoresis, the protein was transferred onto a polyvinylidene difluoride (PVDF) membrane and sequentially detected by primary antibodies, secondary antibodies, and enhanced chemiluminescence. Rabbit anti-PREP (ab58988, Abcam, USA), anti-CD68 (ab125212, Abcam, USA), anti-MMP8 (ab81286, Abcam, USA), anti-MMP9 (ab38898, Abcam, USA), anti-LCN2 (ab216462, Abcam, USA), anti-PERK1/2 (4370T, Cell Signaling Technology, USA), and anti-ERK1/2 (4695T, Cell Signaling Technology, USA) antibodies were used. An anti-GAPDH antibody (AF0006, Beyotime, China) was used for detection of the internal control. The bands were quantified using Image Lab Version 2.0.1 (Bio-Rad, Hercules, CA).
9
Immunohistochemical Analysis of Osteoclasts
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Osteoclasts cultured on dentine or cartilage were fixed in 4% formalin. Dentines were decalcified in 0.5M EDTA prior to paraffin embedding. H&E staining was performed on transverse 5 m sections.
For immunohistochemistry, antigen retrieval of deparaffinised OA tissue sections was performed by immersion in hot citric acid solution. Sections were exposed to primary rabbit monoclonal antibodies against MMP-8 (ab81286, 1:1000, Abcam, Cambridge, UK) or MMP-9 (ab73734, 1:1000, Abcam) overnight at 4C or a PBS control and staining was visualised with DAB. Image acquisition was performed using a Zeiss AxioImager MI microscope, AxioCam HRC camera and AxioVision software.
Osteoclasts in tissue sections were considered as large, multinucleated cells containing ≥3 nuclei; a widely accepted identification criterion [18, (link)22, (link)23] (link).
For immunohistochemistry, antigen retrieval of deparaffinised OA tissue sections was performed by immersion in hot citric acid solution. Sections were exposed to primary rabbit monoclonal antibodies against MMP-8 (ab81286, 1:1000, Abcam, Cambridge, UK) or MMP-9 (ab73734, 1:1000, Abcam) overnight at 4C or a PBS control and staining was visualised with DAB. Image acquisition was performed using a Zeiss AxioImager MI microscope, AxioCam HRC camera and AxioVision software.
Osteoclasts in tissue sections were considered as large, multinucleated cells containing ≥3 nuclei; a widely accepted identification criterion [18, (link)22, (link)23] (link).
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