LPS from Escherichia coli (serotype 0111:B4), diethylenetriamine pentaacetic acid (DTPA), DHR123, 3-morpholinosydnonimine (SIN-1), l -2-amino-3-mercapto-3-methylbutanoic acid (l -penicillamine), and methyl gallate were obtained from Sigma Chemical Co. (St. Louis, MO, USA). DCFH-DA was obtained from Molecular Probes (Eugene, OR, USA), and immobilon-P transfer membranes were purchased from Millipore (Bedford, MA, USA). The antibodies used were sourced as follow: The antibodies to p-ERK, ERK, p-JNK, JNK, p-p38, p38, COX-2, iNOS, IL-6, β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and p-IKKα/β and p65 were from Cell Signaling Biotechnology (Beverly, MA, USA). Enhanced chemiluminescence western blot detection reagents were purchased from Amersham Life Science (Arlington Heights, IL, USA). All chemicals and solvents used were purchased from E. Merck (Frankfurt Str., Darmstadt, Germany), Fluka (St. Louis, Mo, USA) and Sigma-Aldrich Co., unless stated otherwise.
Interleukin 6 (il 6)
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom, Germany, China
IL-6 is a recombinant protein produced by Santa Cruz Biotechnology. It is a cytokine that plays a central role in the immune system and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Santa Cruz Biotechnology (33 mentions)
Il 1β, by Santa Cruz Biotechnology (30 mentions)
β actin, by Santa Cruz Biotechnology (20 mentions)
Il 10, by Santa Cruz Biotechnology (10 mentions)
Cox 2, by Santa Cruz Biotechnology (9 mentions)
Nf κb, by Santa Cruz Biotechnology (8 mentions)
Nf κb p65, by Santa Cruz Biotechnology (7 mentions)
Nf κb p65, by Cell Signaling Technology (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (6 mentions)
Gapdh, by Santa Cruz Biotechnology (6 mentions)
P jnk, by Santa Cruz Biotechnology (5 mentions)
Caspase 1, by Santa Cruz Biotechnology (5 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (5 mentions)
Mmp 9, by Santa Cruz Biotechnology (5 mentions)
Erk1 2, by Cell Signaling Technology (5 mentions)
Bcl 2, by Santa Cruz Biotechnology (5 mentions)
β actin, by Cell Signaling Technology (5 mentions)
Tnf α, by Abcam (5 mentions)
Ifn γ, by Santa Cruz Biotechnology (4 mentions)
116 protocols using interleukin 6 (il 6)
1
LPS-Induced Inflammation Pathway Analysis
2
Immunohistochemical Analysis of Cytokine Expression
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Immunohistochemical analysis was conducted as described in our previous study [9] (link). Briefly, after getting rid of paraffin from tissues on the slides in order to eliminate endogenous peroxidase they were immersed in 3% hydrogen peroxide in methanol for 10 min. Sodium citrate buffer (0.1 M) was used for the antigen retrieval. In order to prevent non-specific binding normal horse serum was used, and then they were bounded with primary antibodies (diluted 1:100 to 1:200) including IFN-γ (Santa Cruz, sc-74104), IL-12p40 (Santa Cruz, sc-57258), IL-4 (Santa Cruz, sc-73318), IL-5 (Santa Cruz, sc-7887), IL-13 (Santa Cruz, sc-1776), TNF-α (BioVision, 3053R-100, Milpitas, CA, USA), and IL-6 (Santa Cruz, sc-1265) antibodies at 4 • C for 1 h. Using biotinylated secondary antibody, all slides were incubated for 10 min and reacted with streptavidin peroxidase complex for 5 min (Vector Laboratories Universal Quick Kit, Burlingame, Canada). Signals were obtained using 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution and they were stained with Mayer's hematoxylin. The results were obtained by an Axioscope A1 (Carl Zeiss) and to measure the positive cells we counted them in five randomly selected non-overlapping fields (×200 magnification) of three separately.
3
Immunohistochemistry for Lung Proteins
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To determine the expression of various target molecules, immunohistochemistry (IHC) was performed. In brief, 5-µm-thick lung sections were deparaffinized and rehydrated through graded concentrations of ethanol. Antigen was retrieved by microwaving in Tris-EDTA buffer (10 mmol/L Tris base, 1 mmol/L EDTA solution, 0.05% Tween-20; pH 9.0). Endogenous peroxidase activity was quenched by incubation in 0.3% hydrogen peroxide (H2O2) solution for 5 minutes. Nonspecific antibody binding was blocked with 1.5% goat or rabbit serum for 30 minutes, and then the sections were incubated with antibodies against interleukin 6 (IL-6; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TGF-β1, and α-smooth muscle actin (α-SMA) (Abcam, Cambridge, MA, USA). Sections were washed with phosphate-buffered saline and processed with secondary antibodies conjugated to HRP-labeled polymer (DakoCytomation EnVision+ System-HRP Labeled Polymer; Dako, Carpinteria, CA, USA), according to the manufacturer's instructions. Peroxidase activity was detected using DAB chromogen substrate solution; finally, the sections were counterstained with hematoxylin.
4
Western Blot Analysis of IL-6 and COX-2
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Protein (60–100 μg per sample) was electrophoresed on 8% or 10% SDS-PAGE and electrophoretically transferred to a PVDF membrane (Amersham Hybond™-P, GE Healthcare, Arlington Heights, IL, USA) at 4 °C for 1.5 h. Unaltered PVDF membranes were stained with Coomassie blue (Bio-Rad, Hercules, CA, USA) to confirm equal protein loading. Blots were incubated for 1 h with 5% nonfat dry milk to block nonspecific binding sites and subsequently incubated at 4 °C overnight with a 1:200 to 1:1000 dilution of polyclonal/monoclonal antibodies against IL-6 (Santa Cruz Biotechnology, Dallas, TX, USA) and COX-2 (Abcam, Cambridge, UK). Immunoreactivity was detected using a peroxidase-conjugated antibody and visualized by the ECL Plus Western Blotting Detection System (#GERPN2232, GE Healthcare, Arlington Heights, IL, USA). The blots were stripped before re-probing with an antibody against GAPDH (Abcam, Cambridge, UK). Densitometry of the bands was measured and normalized with GAPDH using NIH ImageJ 1.5.0 (64-bit) software.
5
Histological Analysis of Rat Joint Tissues
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Rat joint tissue was fixed in 4% paraformaldehyde, decalcified in EDTA bone decalcifier and embedded in paraffin. The sections (7 μm) were stained with hematoxylin and eosin (H&E) and toluidine blue to detect proteoglycans. For immunohistochemistry, rat joint sections were blocked with 1% normal goat serum and stained with antibodies to IL-6 (1 μg/mL, Santa Cruz, CA, USA), COX-II (200 ng/mL, Santa Cruz, CA, USA) and isotype control antibody (1 μg/mL, Santa Cruz, CA, USA) at 4 °C overnight. After three washes in PBS, the secondary antibody (biotin-labeled goat anti-rabbit IgG) was applied for 1 h at room temperature. Staining was detected with 3, 3′-diaminobenzidine tetrahydrochloride and the sections were then counterstained with H&E and observed under a light microscope.
6
Immunofluorescence Staining Protocol for Tissue Slices
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The immunofluorescence staining protocol followed was according to the study of Zaquot and colleagues [55 (link)] with minor modifications. All tissues slices were deparaffinized using xylene, ethanol (100%, 95%, 90%, 80%, and 70%), and PBS. After that, for the heat antigen retrieval (unmasking) step, an antigen retrieval buffer (pH 6.0) containing 10% FBS in PBS and citrate buffer was used to microwave-boil all slices for 20 min. For the blocking step (about 1 h), 4% BSA in PBS was added to the slices.
Next, for the permeabilization step, tissue slices were incubated with primary antibodies against α-SMA, vimentin, IL-1β, IL-6, IL-8, TNF-α, MCP-1, and CD68 (Santa Cruz Biotechnology; 1:100) overnight at 4 °C. The second antibody, Alexa fluor 488 goat anti-mouse IgG, was stained on the slices 1 h after three PBS washes. Finally, all tissues were mounted with 4′,6-diamidino-2-phenylindole (DAPI VECTASHIELD®) (Vector Laboratories, Burlingame, CA, USA) for fluorescence imaging and kept at −20 °C.
Next, for the permeabilization step, tissue slices were incubated with primary antibodies against α-SMA, vimentin, IL-1β, IL-6, IL-8, TNF-α, MCP-1, and CD68 (Santa Cruz Biotechnology; 1:100) overnight at 4 °C. The second antibody, Alexa fluor 488 goat anti-mouse IgG, was stained on the slices 1 h after three PBS washes. Finally, all tissues were mounted with 4′,6-diamidino-2-phenylindole (DAPI VECTASHIELD®) (Vector Laboratories, Burlingame, CA, USA) for fluorescence imaging and kept at −20 °C.
7
Cytokine and Signaling Pathway Analysis
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Roswell Park Memorial Institute medium (RPMI 1640), fetal bovine serum (FBS), and antibiotics were purchased from GIBCO BRL (Grand Island, NY). Culture plastic wares were obtained from NUNC (Roskilde, Denmark). Silica gel 60 HPTLC plates used were from E. Merck (Darmstadt, Germany). The TNF-α assay kit was procured from Amersham (NJ, USA) and PGE-2 kit from R & D system (MN, USA). IL-1β, IL-6, IL-10, IL-12p40, IL-17, and BD OptEIA assay kits were from BD Biosciences (USA), nitric oxide assay kit was from Calbiochem (Darmstadt, Germany), and goat anti-TNF-α, -IL-1β, -IL-6, -IL-10, -NF-κB p65, -IκB, -histone-H2B, -β-actin, -COX-2, -iNOS, -ICAM-1, -VCAM-1, -E-Selectin, -P-Selectin, and rabbit anti-CD14, -TLR4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
8
Histological Analysis of Joint Inflammation
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Mouse joint tissue was fixed in 4% paraformaldehyde, decalcification EDTA bone decalcifier and embedded in paraffin. The section (7 μm) was stained with hematoxylin and eosin, Safranin O, and toluidine blue to detect proteoglycans.
Immunohistochemistry was performed using the Vectastatin ABC kit (Vector Laboratories, Burlingame, CA, USA). Tissue was first incubated with primary antibodies to IL-17, IL-6, IL-1b, TNF-α, Nitrotyrosine, NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HO-1, Inos (Abcam, Cambridge Science Park, Cambridge, UK) overnight at 4°C. The sections were counterstained with hematoxylin. Samples were photographed with an Olympus photomicroscope (Tokyo, Japan).
Immunohistochemistry was performed using the Vectastatin ABC kit (Vector Laboratories, Burlingame, CA, USA). Tissue was first incubated with primary antibodies to IL-17, IL-6, IL-1b, TNF-α, Nitrotyrosine, NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HO-1, Inos (Abcam, Cambridge Science Park, Cambridge, UK) overnight at 4°C. The sections were counterstained with hematoxylin. Samples were photographed with an Olympus photomicroscope (Tokyo, Japan).
9
Diosmetin Regulation of Inflammatory Pathways
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Diosmetin was bought from ChemFaces Bioschemical Co., Ltd. (Wuhan, China). L-NAME and captopril were supplied by Sigma-Aldrich Corp. (St Louis, MO, USA). This study primarily used mouse antibodies to Nrf2, HO-1, IL-6 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and p-JNK and the rabbit primary antibody to p-NF-κB (Cell Signaling Technology, Inc., Danvers, MA, USA). A second antibody, goat anti-mouse antibody (Abcam Plc, Cambridge, UK), was used in this study. The intensity of β-actin protein expression was used as a control protein.
10
Forskolin Regulation of HeLa Cell Apoptosis
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HeLa cells were commercially sourced from NCCS (National Centre for Cell Science, Pune, India) in mycoplasma free condition. DMEM with high glucose, foetal bovine serum (FBS) and PBS from Gibco BRL (Carlsbad, CA, USA). The chemicals and reagents were purchased from Merck, nitro cellulose membrane was obtained from Millipore (Bangalore, India). The antibodies Bax, Bcl2, Caspase-3 were sourced from Cell Signalling Technologies, PP2B, IL-6 and NOS2 primary antibodies are purchased from Santa Cruz biotechnologies (Dallas, TX, USA). Correspondingly, Forskolin purchased from sigma (St. Louis, MO, USA) and the 10 mM stock of Forskolin (M.W. 410.5) was prepared with DMSO and all sub stocks and working stocks were prepared with 1 × PBS.
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