Reagents used for cell culture were purchased from GIBCO (Invitrogen Corporation, Carlsbad, CA). Anti-RAGE-blocking antibody used in animals was purchased from RD System (Minneapolis, MN). LPS was purchased from Sigma–Aldrich (St. Louis, MO, USA); recombinant HMGB1 protein was purchased from Novus Biologicals (Colorado, USA). Anti-nestin antibody was purchased from Cell Signaling (Danvers, MA). Anti-RAGE antibody, anti-MAP-2 antibody, anti-SOX-2 antibody, and Tnf-a and IL-1β ELISA kits were purchased from Abcam (Cambridge, UK).
Anti rage antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-RAGE antibody is a protein that binds to the receptor for advanced glycation end products (RAGE). RAGE is a cell surface receptor involved in various cellular processes. This antibody can be used to detect and study the expression and role of RAGE in biological samples.
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Lab products found in correlation
Anti hmgb1 antibody, by Abcam (2 mentions)
Anti occludin antibody, by Abcam (2 mentions)
Anti nf kb p65 antibody, by Abcam (2 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Abcam (2 mentions)
Anti zo 1 antibody, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti gapdh antibody, by Abcam (2 mentions)
Anti sox2 antibody, by Abcam (1 mentions)
Anti rage blocking antibody, by R&D Systems (1 mentions)
Anti map2 antibody, by Abcam (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Fitc conjugated donkey anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
Vectashield dapi, by Vector Laboratories (1 mentions)
Tcs sp8, by Leica (1 mentions)
Ix70 fluorescence microscope, by Olympus (1 mentions)
Hepes, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti tlr2 fitc, by BioLegend (1 mentions)
12 protocols using anti rage antibody
1
Reagents and Antibodies for Cell Culture and Analysis
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Immunofluorescence analysis of RAGE and MAPK
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THP-1 Macrophage Model for AGEs and oxLDL
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Human monocytic cell line THP-1 was purchased from Scientific Research Institute, Shanghai, China and cultured in RPMI 1640 medium containing 10 % FBS, 10 mM HEPES (Sigma, USA), and 1 % pen/strep solution at a density of 5 × 105cells/ml in a 5 % CO2 incubator. The cells were seeded in six-well plates for 48 h in the presence of 100 ng/ml PMA, (Sigma, USA) which allowed them to differentiate into adherent macrophages. The culture medium was then changed into RPMI1640 medium containing 0.1 % FBS for 6 h of cell starvation. The macrophages were pretreated with BSA (600 μg/ml) as control group or with different concentration of AGEs (300 μg/ml and 600 μg/ml) for 2 h, then stimulated with oxLDL (100 μg/ml) (Jingke Chemistry, China) for 24 h at 4 °C in a 5 % CO2 incubator, and then collected for detection. To observe the effect of RAGE, one group of cells was treated with anti-RAGE antibody (10 μg/ml)(Abcam, USA) for 2 h before adding high concentration of AGEs (600 μg/ml).
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Multiparametric Immunophenotyping of Cells
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HMGB1 and RAGE Protein Quantification in Lung Tissues
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The protein levels of HMGB1 and RAGE in lung tissues were measured by the western blot analysis as described previously [27 (link), 28 ]. Briefly, soluble protein extracts (20 μg) from the lung tissues were subject to 8% SDS-polyacrylamide gels and then transferred to polyvinyldifluoride (PVDF) membranes. After blocking in nonfat milk, the membranes were exposed to primary anti-HMGB1 antibody (1 : 1000 dilution; Abcam, Hong Kong, China) or anti-RAGE antibody (1 : 1000 dilution; Abcam, Hong Kong, China) overnight at 4°C. After incubation with horseradish peroxidase-linked anti-rabbit secondary antibodies, the proteins were detected with enhanced chemiluminescence reagent and finally exposed to X-ray films for the detection of target proteins.
6
Histological Analysis of Dupuytren's Disease
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HE staining was performed to observe the histological differences between the control group and DD group. The sections were incubated with alpha-smooth muscle actin (alpha-SMA) antibody (Sigma-Aldrich, St Louis, MO) to confirm the expression of myofibroblasts that characterize DD. The sections were also incubated at 4 °C overnight with the following primary antibodies: anti-AGEs antibody (10 μg/ml; Abcam, Cambridge, UK), anti-RAGE antibody (10 μg/ml; Abcam), and anti-reactive oxygen species modulator 1 (ROMO1) antibody (0.7 μg/ml; Abcam). After incubation with the primary antibodies, the sections were incubated with the secondary antibody, Alexa Fluor 594 (20 μg/ml; Abcam), and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Digital images of AGEs, RAGE, ROMO1 (red), and nuclear (blue) staining were captured by using a Bio Zero BZ-8000 (Keyence, Osaka, Japan). The positive areas were quantified by using selective coloring in Adobe Photoshop (CS6; Adobe Systems, San Jose, CA, USA) [13 (link)].
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Western Blot Analysis of Tight Junction and Inflammatory Proteins
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Total proteins of cells were obtained by RIPA Lysis buffer (Beyotime Biotechnology, Jiangsu, China). The proteins were separated by 10% SDS-PAGE, transferred to polyvinylidene uoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA), and blocked with TBST containing 1.5% BSA at 26 ℃. After 2 h, the membranes were incubated with anti-ZO-1 antibody (1:1000, Invitrogen), anti-Occludin antibody (1:2000, Abcam), anti-RAGE antibody (1:500, Abcam), anti-NF-kB p65 antibody (1:1000, Abcam) and anti-GAPDH antibody (1:4000, Abcam), respectively, at 4°C overnight. After washed three times, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:500, Abcam) at 37 ℃ for 2 h. The protein bands were visualized with ECL kit (Thermo Fisher Scienti c, Rockville, MD, USA), and images were taken with image lab software (Bio-Rad, Hercules, CA, USA).
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AGEs-mediated Cardiomyocyte Regulation
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Neonatal cardiomyocyte were treated with AGEs (10 mg/L, 25 mg/L, 50 mg/L and 100 mg/L for 48 hours). Also, anti-RAGE antibody (10 μg/ml, Abcam, USA) was used to block the binding of AGEs to RAGE. An mTOR inhibitor, Rapamycin (100 nM, Invitrogen, USA) was added to cultured cells to block the PI3K/Akt/mTOR in this study.
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Immunohistochemical Analysis of RAGE and CD31
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Deparaffinized sections were boiled in 1× target retrieval solution (Dako, Capenteria, CA) for 20 min. Endogenous peroxidase activity was quenched by treating samples with 3% hydrogen peroxide for 5 min. After blocking in 5% normal goat serum diluted in PBS (1X; 155 mM NaCl, 1.06 mM KH2PO4, 2.97 mM Na2HPO4-7H2O, pH 7.4), the sections were incubated with anti-RAGE antibody (1:125, Abcam, Cambridge, MA), anti-CD31 antibody (1:100, Abcam), or the isotype- and concentration-matched normal immunoglobulin G (IgG) control (Abcam) overnight at 4 °C. After three washes with PBST (PBS plus 0.1% Tween-20), the sections were then incubated with the secondary biotinylated goat anti-rabbit or anti-mouse IgG (1:2,000) for 1 h at room temperature. Sections were then detected with the alkaline phosphatase detection system (Vectastain ABC-AP kit, Vector Laboratories, Burlingame, CA), and a blue reaction product was produced by incubating sections with the alkaline phosphatase substrate (Vector Blue AP Substrate Kit III, Vector Laboratories). Sections were also counterstained with Nuclear Fast Red (Vector Laboratories).
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Western Blot Analysis of Tight Junction and Inflammatory Proteins
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Total proteins of cells were obtained by RIPA Lysis buffer (Beyotime Biotechnology, Jiangsu, China). The proteins were separated by 10% SDS-PAGE, transferred to polyvinylidene uoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA), and blocked with TBST containing 1.5% BSA at 26 ℃. After 2 h, the membranes were incubated with anti-ZO-1 antibody (1:1000, Invitrogen), anti-Occludin antibody (1:2000, Abcam), anti-RAGE antibody (1:500, Abcam), anti-NF-kB p65 antibody (1:1000, Abcam) and anti-GAPDH antibody (1:4000, Abcam), respectively, at 4°C overnight. After washed three times, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:500, Abcam) at 37 ℃ for 2 h. The protein bands were visualized with ECL kit (Thermo Fisher Scienti c, Rockville, MD, USA), and images were taken with image lab software (Bio-Rad, Hercules, CA, USA).
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