Formalin-fixed and paraffin-embedded tumor and spheroid sections (3 µm) were dewaxed using Roticlear (Carl Roth, Karlsruhe, Germany) and rehydrated in a graded series of ethanol. Antigen retrieval was performed in 10 mmol/L citrate buffer pH 6 intermittently heated to 100 °C in 5 min intervals. Washing was performed using 0.05 mol/L Tris-buffered saline pH 8 containing 0.5% (v/v) Tween-20 (TBS-T). Endogenous peroxidase was quenched using 3% H2O2 in TBS-T. Endogenous avidin and biotin were blocked using a commercially available avidin/biotin quenching system (Agilent, Santa Clara, CA, USA). Non-specific binding sites were blocked using 10% fetal bovine serum (v/v) in TBS-T. COX-2 was detected using the primary antibody ab15191 (Abcam, Cambridge, UK). Isotype controls were incubated with non-specific rabbit IgG ab27478 (Abcam). Specific binding was detected using the biotinylated secondary antibody 111-065-003 (Dianova, Hamburg, Germany) and ExtrAvidin-peroxidase E2886 (Sigma-Aldrich, St. Louis, MO, USA) followed by staining with 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich). Tumor sections were counterstained with Meyer’s hematoxylin, mounted with Kaiser’s glycerol gelatin (Carl Roth), and imaged using the AXIO Imager A1 microscope (Carl Zeiss, Oberkochen, Germany).
Ab15191
Manufactured by Abcam
Sourced in United States, United Kingdom, Japan
Ab15191 is a Rabbit polyclonal antibody targeting ABCG2. This antibody is suitable for use in Western Blot and Immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab15323, by Abcam (8 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (5 mentions)
Ab3523, by Abcam (4 mentions)
Ab125066, by Abcam (4 mentions)
Ab205718, by Abcam (3 mentions)
Ab41037, by Abcam (3 mentions)
Ab9324, by Abcam (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab16502, by Abcam (3 mentions)
Ab8226, by Abcam (3 mentions)
Ab97779, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Exoab cd63a 1, by System Biosciences (3 mentions)
Ab16663, by Abcam (2 mentions)
Ab207297, by Abcam (2 mentions)
Ab9056, by Abcam (2 mentions)
Ab188570, by Abcam (2 mentions)
Ab3773, by Abcam (2 mentions)
94 protocols using ab15191
1
Immunohistochemical Analysis of COX-2 in Tumor Sections
2
Protein Expression Analysis in H9c2 Cells
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Proteins in H9c2 cells were extracted by lysis buffer (P0013J, Beyotime). After determining the concentration with a BCA detection kit (P0012, Beyotime), the proteins were loaded and electrophoresed on 10% SDS polyacrylamide gel (P0012AC, Beyotime), and then transferred onto PVDF membranes (FFP26, Beyotime,). After using 5% non-fat milk to block membranes for 60 min at 37°C, the following primary antibodies were incubated with membranes overnight at 4°C: Bcl-2 (ab59348, 1:1000, 26 kDa, Abcam, USA), Bax (ab32503, 1:1000, 21 kDa, Abcam), Cleaved caspase-3 (ab49822, 1:500, 17 kDa, Abcam), iNOS (ab3523, 1:200, 135 kDa, Abcam), Cox-2 (ab15191, 1:250, 69 kDa, Abcam), CDK4 (ab199728, 1:2000, 34 kDa, Abcam), Cyclin D1 (ab16663, 1:25, 36 kDa, Abcam), HSP70 (ab181606, 1:1000, 70 kDa, Abcam), and GAPDH (ab181602, 1:10000, 36 kDa, Abcam). Following extensive washing, protein bands were incubated with the secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab205718, 1:2000, 42 kDa, Abcam) at 37°C for 2 h. The detection of signal was performed according to a standard ECL method (27), and analysis software (Image J 1.5i, National Institutes of Health, USA) was used for images to measured protein expression. GAPDH was used as housekeeping gene.
3
Quantifying Hippocampal COX-II Protein Levels
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Proteins in the hippocampal tissue homogenates were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and incubated with the anti‐COX‐II primary antibody (ab15191; Abcam) for 12 hr, followed by the horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (A0545‐1ML; Sigma‐Aldrich) for 1 hr. Enhanced chemiluminescence was used to determine protein levels of COX‐II (Nie et al., 2017 ).
4
Western and Isoelectric Blotting of Brain Proteins
5
Immunohistochemical Analysis of COX-2 and Survivin
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Sections from formalin-fixed, paraffin-embedded tumor tissues (4-µm thick) were immunostained and assayed using a PV6000 polymer system (ZSGB-BIO, Beijing, China). All paraffin sections were heated to 90°C for 20 min. Following routine deparaffinization by serial baths in xylene 3 times and dehydration in a graded series of ethanol (100, 95 and 80%) and distilled water, each section was treated with 0.3% hydrogen peroxide to block endogenous peroxidase activity. Sections were subsequently washed in 0.01 M phosphate-buffered saline (PBS; pH 7.4), microwaved at 100°C for 20 min for antigen retrieval and then incubated overnight at 4°C with optimal dilutions of primary antibodies. Antibodies used were anti-COX-2 rabbit polyclonal antibody (dilution, 1:400; ab15191; Abcam, Cambridge, UK) and anti-survivin rabbit polyclonal antibody (dilution, 1:1,200; ab24479; Abcam). Following washing in PBS, the tissue sections were treated with secondary antibody, IgG-horseradish peroxidase polymer multimer (PV-6000; ZSGB-BIO, Beijing, China), at 37°C for 20 min. Finally, the tissue sections were incubated with diaminobenzidine and counterstained with hematoxylin, cleared and mounted on neutralbalsam. In order to confirm COX-2 and survivin immunospecificity, 0.01 M PBS was supplied instead of primary antibody, as the negative control.
6
Western Blot Analysis of Chondrocyte Signaling
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Cells were lysed on ice for 30 min in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml aprotinin). Protein fractions were collected by centrifugation at 15 000 × g at 4 °C for 10 min, subjected to 10% SDS-PAGE, and then electrotransferred onto nitrocellulose membranes (Whatman, Piscataway, NJ, USA). The membranes were blocked with 5% BSA and then incubated with specific antibodies overnight at 4 °C. The primary antibodies were from the following sources: Sox-9 (1 : 500, ab26414; Abcam), Col2a1 (1 : 1000, BS1071; Bioworld Technology, St. Louis Park, MN, USA), MMP-3 (1 : 200; sc-6839; Santa Cruz), MMP-13 (1 : 200; sc-30073; Santa Cruz), Adamts-5 (1 : 200; sc-83186; Santa Cruz), COX-2 (1 : 500, ab15191; Abcam), JNK, phospho-JNK, Erk, phosphor-Erk, p38, phospho-p38, p65, phospho-p65, IκBα, phospho-c-Jun, phosphor-ATF2, MKK4, phospho-MKK4, HGK (1 : 1000; all from Cell Signaling Technology), and GAPDH (1 : 5000, G9545 Sigma-Aldrich, St. Louis, MO, USA). HRP-conjugated secondary antibodies (Cell Signaling Technology) were used at a 1:1000 dilution. The antigen–antibody complexes were visualized using the enhanced chemiluminescence detection system (Millipore, Darmstadt, Germany) as recommended by the manufacturer.
7
Inflammatory Protein Expression in Hippocampus
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Hippocampal tissues were isolated from mice of each group (n = 10) and then lysed with RIPA buffer containing protein phosphatase inhibitor (Sigma-Aldrich). Total proteins were extracted and transferred onto a PVDF membrane, followed by blockage for 2 h, and incubation with primary antibodies [IL-17A antibody (ab9056), iNOS antibody (ab15323), NF-κB p65 antibody (ab207297), and COX-2 antibody (ab15191)(all 1:1,000; Abcam) and secondary antibodies. In this study, β-actin served as the internal reference protein. The ultra-sensitive chemiluminescent liquid-based FujiFilm LAS 4000 imaging analyzer (FujiFilm, Tokyo, Japan) was used for visualization, and Image J (NIH, Bethesda, MD) was used to analyze the relative intensities of individual bands.
8
Western Blot Analysis of Protein Expression
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After treatment, cells were harvested in a mammalian protein extraction reagent buffer (Thermo Fisher Scientific, IL, USA) on ice. Total proteins were resolved on 8–12% SDS-PAGE gels and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in TBST and then incubated overnight at 4 °C with the first antibodies against IL-6 (Abcam, ab9324, 1:1000), COX2 (Abcam, ab15191, 1:1000), aggrecan (Abcam, ab3773, 1:200), collagen II (Abcam, ab188570, 1:2000), MMP3 (Abcam, ab53015, 1:1000), ATAMTS5 (Abcam, ab41037, 1:250), FBXO6 (Abcam, ab153853, 1:1000) and β-Tubulin (Servicebio, GB11017B, 1; 1000). After washing membranes three times using TBST, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (YEASEN, Shanghai, China). After washing, immunolabeling was detected using the ECL reagent (Thermo Fisher Scientific, IL, USA).
9
Western Blot Analysis of Lipid-Binding Proteins
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Western blot experiments were performed as described [5 (link)]. Blots were probed with the following antibodies: COX2 (1:1000, Abcam #Ab15191), FAAH (1:1000, Abcam #Ab54615), MAGL (1:400, Cayman Chemical #10212), GAPDH (1:5000, Abcam #Ab8245), CB1 (1:1000, Abcam #Ab172970), NAPE-PLD (1:400, Abcam #Ab95397), FABP5 (1:1000, BioVendor R&D #RD181060100), or FABP7 (1:200, Abcam #Ab32423). The blots were developed using the Immun-star HRP substrate (Bio-Rad) and scanned using a C-DiGiT scanner (Li-COR). Protein band intensities were quantified and normalized to the respective GAPDH intensities. To quantify changes in protein expression between WT and FABP5/7 KO mice, ratios of normalized protein intensities were compared between the genotypes.
10
RKC-B1 Modulates Inflammatory Pathways
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RKC-B1 was kindly provided by Prof. Jian Zhou (Fujian Institute of Microbiology) and Prof. Hua-Wei Zhang (Zhejiang University of Technology). LPS (Escherichia coli 0127: B8) were purchased from Sigma Aldrich (St. Louis, MO, USA). ELISA kits IL-1β (EM001), IL-6 (EM004), MCP-1 (EM018) and ICAM-1 (EM013) were purchased from ExCell Biology (Shanghai, China). Antibodies for COX2 (ab15191) and IL-18 (ab207323) were purchased from Abcam (Cambridge, UK). Antibodies for β-actin (3700), NF-κB p65 (8242), phospho-NF-κB p65 (3033), IκBα (4814), phospho-IκBα (2859), NLRP3 (15101) and cleaved caspase-1 (89332) were purchased from Cell Signaling Technology (Beverley, CA, USA).
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