Mouse corneas were excised at 0, 2, 4, 6, and 24 hours post-wounding and immediately flash frozen in liquid nitrogen. Corneas were thawed in 200 µl of buffer containing 20-mM Tris-HCl, 150-mM sodium chloride, 1-mM phenylmethylsulfonylfluoride, 0.05% Tween 20, and a 1X Halt Protease Inhibitor Cocktail from Thermo Fisher Scientific. Homogenates were created by disrupting the corneas for 10 minutes at maximum speed in a Bullet Blender (Next Advance, Inc., Averill Park, NY, USA) using 0.9- to 2-mm stainless steel beads. Homogenates were centrifuged at 16,000g for 10 minutes to eliminate the froth. Protein concentrations of lysates were determined. Equal amounts of protein (20 µg) from each sample of corneal homogenate were analyzed by electrophoresis on a 12% SDS-PAGE gel and transferred to nitrocellulose membranes (Whatman, Inc., Florham Park, NJ, USA) for western blot analysis. Blots were treated as described previously to quantify S100A9 and β-actin. The mouse monoclonal anti-β-actin primary antibody was from MilliporeSigma (Burlington, MA, USA) and the rabbit polyclonal anti-S100A9 was from Abcam. Blots were analyzed and semiquantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Anti s100a9
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-S100A9 is a primary antibody that detects the S100A9 protein. S100A9 is a calcium-binding protein involved in the regulation of inflammatory processes and immune response. This antibody can be used for various research applications, such as Western blotting, immunohistochemistry, and flow cytometry.
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Lab products found in correlation
Bullet blender, by Next Advance (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
Anti hla dr, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti p p65, by Cell Signaling Technology (1 mentions)
Anti cd8, by BD (1 mentions)
Bay 11 7082, by Beyotime (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti phospho p p38, by Cell Signaling Technology (1 mentions)
Anti cd33, by Thermo Fisher Scientific (1 mentions)
Anti rage, by Santa Cruz Biotechnology (1 mentions)
Anti p jnk, by Cell Signaling Technology (1 mentions)
Anti tlr4, by Santa Cruz Biotechnology (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
9 protocols using anti s100a9
1
Quantifying S100A9 Expression in Wounded Mouse Corneas
2
Molecular Mechanisms in Inflammatory Signaling
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The antibodies included anti-S100A9 (Cat no. ab92507; Abcam), anti-TLR4 (Cat no. sc-293072; Santa Cruz Biotechnology), anti-RAGE (Cat no. sc-80653; Santa Cruz Biotechnology), anti-p38 (Cat no. 9212; Cell Signaling Technology), anti-p65 (Cat no. 3034; Cell Signaling Technology), anti-ERK1/2 (Cat no. 4695; Cell Signaling Technology), anti-JNK (Cat no. 9253; Cell Signaling Technology), anti-AKT (Cat no. 8596; Cell Signaling Technology), anti-phospho(p)-p38 (Cat no. 4511; Cell Signaling Technology), anti-p-p65 (Cat no. 3033; Cell Signaling Technology), anti-p-ERK1/2 (Cat no. 3510; Cell Signaling Technology), anti-p-JNK (Cat no. 4668; Cell Signaling Technology), anti-p-AKT (Cat no. 9271; Cell Signaling Technology), anti-CD8 (Cat no. 340046, BD), anti-HLA-DR (Cat no. 4310370, eBioscience), anti-CD33 (Cat no. 4296343, eBioscience) and CD11b (Cat no. 4291932, eBioscience), and horseradish peroxidase-conjugated anti-mouse, anti-rabbit IgG antibodies. The inhibitors contained TAK-242 (MedChemExpress, New Jersey), FPS-ZM1 (MedChemExpress, New Jersey), SB203580 (Beyotime) and BAY 11-7082 (Beyotime). The preparation of the recombination GST-S100A9 protein, as well as its control protein GST, have previously been described (21 (link)).
3
Antibody panel for cell analysis
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The following antibodies were used in this study: anti-CD11b-FITC (Biolegend, 101206, dilution ratio: 1:100 for flow cytometry and immunofluorescence); anti-Ly-6G-PE (eBioscience, 12-9668-82, dilution ratio: 1:100 for flow cytometry); anti-Ly-6C-PE (Cell eBioscience, 12-5932-82, dilution ratio: 1:100 for flow cytometry); anti-p-Smad1/5 (Cell Signaling Technology, 9516, dilution ratio: 1:100 for immunofluorescence and 1:1000 for Western blot); anti-Smad1/5 (Cell Signaling Technology, 6,944, dilution ratio: 1:100 for immunofluorescence and 1:1000 for Western blot); anti-p-Stat3 (Cell Signaling Technology, 9,145, dilution ratio: 1:1000 for Western blot); anti-Stat3 (Cell Signaling Technology, 9,139, dilution ratio: 1:1000 for Western blot); anti-p-Erk (Cell Signaling Technology, 4,370, dilution ratio: 1:1000 for Western blot); anti-Erk (Cell Signaling Technology, 4,695, dilution ratio: 1:1000 for Western blot); anti-mouse IL6 (Biolegend, 504501, dilution ratio: 1:500 for neutralization); anti-S100A9 (Abcam, GB111149, dilution ratio: 1:2000 for IHC); anti-Ki67 (Servicebio, GB13030-2, dilution ratio: 1:500 for IHC), and anti-β-actin (Sigma, A1978, dilution ratio: 1:1000 for Western blot). Rat tail collagen I (Corning, 356236) and RhBMP2 (R&D, 355-BM-100) were also used. Tris–HCl, NaCl, and other chemicals were from Sigma.
4
Quantifying Renal Immune Markers
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The expression levels of S100A9,F4/80, ki67 on the renal sections were determined using immunohistochemistry as previously described (Chen et al., 2022 (link)). These expression levels were determined with anti-S100A9 (Abcam, United Kingdom, 1:500), anti-F4/80 antibody (Abcam, United Kingdom, 1:100) or anti-ki67 (Abcam, United Kingdom, 1:300). After the slices were dewaxed, they were incubated with anti-S100A9, anti-F4/80 antibody or anti-ki67 overnight at 4°C. Then, the color reaction was performed using a diaminobenzidine (DAB) coloring kit (Gene Tech, Shanghai, China). All images were randomly selected from 5 visual fields for each slice and photographed with a Nikon microscope (Nikon, Tokyo).
5
Immunohistochemical Analysis of Skin Markers
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The 4% formaldehyde-fixed and dehydrated skin tissue samples were embedded in paraffin wax and cut into 5-µm-thick sections. The skin sections were incubated with a primary antibody against human KRT10 (1:100; Abcam, Cambridge, UK), followed by incubation with the adequate secondary antibody. After staining, sections were counterstained with hematoxylin to provide contrast. For immunofluorescence staining, the skin tissues were quench-frozen and embedded in OCT. The cryosections (10 µm) were fixed in chilled acetone for 15 min and incubated overnight at 4 °C with anti-S100A9 (1:400; Abcam, Cambridge, UK), anti-HBD-2 (1:400; Abcam, Cambridge, UK), anti-Filaggrin (1:400; Abcam, Cambridge, UK), anti-Involucrin (1:400; Abcam, Cambridge, UK), anti-AO-1 (1:400; Abcam, Cambridge, UK), anti-CLDN-1 (1:400; Abcam, Cambridge, UK), anti-KRT6 (1:400; Abcam, Cambridge, UK), anti-KRT10 (1:400; Abcam, Cambridge, UK), and anti-KRT17 (1:400; Abcam, Cambridge, UK) primary antibodies, followed by Alexa Fluor 488- or 568-conjugated secondary antibodies and DAPI. The immunostained images were obtained using a digital camera (DP74; Olympus, Tokyo, Japan) coupled with an optical microscope (BX53; Olympus, Tokyo, Japan).
6
Kidney Immune Cell Profiling
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For the double immunostaining of kidney tissue, anti-S100A9 (Abcam, United States, 1:1000) and anti-F4/80 (Abacam, 1:400) were used to stain kidney tissue section at 48 h of CLP surgery.
7
Histopathological Analysis of Oral Tissues
8
Protein Expression Profiling in Cells
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Cells were lysed with lysis buffer containing a protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA). We used anti-S100A9 (Abcam for humans and R&D for mice), anti-phospho-S100A9 (ThermoFisher Scientific), anti-phospho-ERK (Abcam), anti-ERK (Abcam), anti-phospho-p38 (Abcam), anti-p38 (Abcam), anti-phospho-NF-κB (Abcam), anti-NF-κB (Cell Signaling Technology), anti-phospho‐Smad 3 (Abcam), anti-Smad 3 (Abcam), anti-MPO (Abcam), anti-NE (Santa Cruz Biotechnology), anti-CD68 (Abcam), anti-inducible nitric oxide synthase (iNOS) (Abcam), and anti-Arginase 1 (ThermoFisher Scientific) antibodies.
9
Immunohistochemical Analysis of CD33 and S100A9
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Immunohistochemical staining for CD33 and S100A9 was performed using anti-human CD33 (Cat no. ab92507, eBioscience) and an anti-S100A9 (Cat no. ab92507, Abcam) antibodies following the manufacturer's instructions. Briefy, the deparaffinized and dehydrated sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 0.3% hydrogen peroxide (H2O2) in methanol for 15 min to block endogenous peroxidase, incubated with primary and peroxidase-tagged secondary antibodies sequentially, and colorized with 0.05% 3,3-diaminobenzidine tetrachloride (DAB). The sections were counterstained with hematoxylin, and observed, and representative images were captured under an inverted phase contrast microscope (Olympus B640, Japan).
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