To detect the expression of IL-6 and PGRN in normal and HCC tissue, the human liver carcinoma and normal tissue microarrays (BC03119a, US Biomax, Inc., Rockville, MD) containing 95 HCC and 10 normal liver tissues were examined by immunohistochemical staining. This part of the study did not require approval from the Institutional Review Board as the tissue microarray was commercially sourced. Immunohistochemistry was performed as described59 (link) with IL-6 antibody (1:400, Abcam, Cambridge, UK) and PGRN antibody (1:50, Santa Cruz Biotechnology, Dallas, TX). Semi-quantitative analysis of the staining intensity score was as described22 (link).
Il 6 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
The IL-6 antibody is a laboratory tool used to detect and quantify the presence of the interleukin-6 (IL-6) cytokine in biological samples. IL-6 is a signaling protein involved in various immune and inflammatory processes. This antibody can be used in techniques such as ELISA, Western blotting, and immunohistochemistry to measure IL-6 levels.
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Lab products found in correlation
Tnf α antibody, by Abcam (3 mentions)
Pgrn antibody, by Santa Cruz Biotechnology (1 mentions)
P stat3 antibody, by Cell Signaling Technology (1 mentions)
P53 antibody, by Cell Signaling Technology (1 mentions)
β actin antibody, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg antibody, by ABclonal (1 mentions)
Apomorphine, by Merck Group (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
6 hydroxydopamine, by Merck Group (1 mentions)
Emodin, by Chengdu Must Bio-Technology (1 mentions)
Alexa fluor 555 labeled donkey anti mouse igg h l, by Beyotime (1 mentions)
2 3 5 triphenyltetrazolium chloride, by Merck Group (1 mentions)
Anti gapdh, by Affinity Biosciences (1 mentions)
Talent qpcr premix sybr green, by Tiangen Biotech (1 mentions)
Anti occludin, by Abcam (1 mentions)
Vegfa antibody, by Affinity Biosciences (1 mentions)
17 protocols using il 6 antibody
1
Immunostaining of IL-6 and PGRN in HCC
2
Immunohistochemical Analysis of IL-6 and pSTAT3
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TMA sections were deparaffinized and rehydrated using xylene and graded concentrations of ethanol. Heat-induced antigen retrieval was performed in citrate buffer (10 mM citrate, pH 6.0). Endogenous peroxidase was blocked in a 0.3% H2O2 solution, after which sections were incubated with primary antibodies overnight at 4°C; rabbit polyclonal IL-6 antibody (1:400, Abcam), rabbit polyclonal to IL-6Rα (1:800, Santa Cruz Biotechnology), and rabbit monoclonal pSTAT3 antibody (1:150, Cell Signaling Technology, clone Tyr705). The antibodies were detected using HRP-labeled secondary (goat anti-rabbit) and tertiary (rabbit anti-goat) antibodies for 30 min at RT (1:100, DAKO,), and visualized with 3,3-diaminobenzidine. Hematoxylin was used for counterstaining.
3
Quantification of Protein Expression in Chondrocytes
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The expression level of the target protein in chondrocytes of each group was calculated by Western blot and semi-quantitative analysis, using methods we described previously [31 (link)]. The following antibodies were used: TNF-α antibody (1: 1000; Abcam, Cambridge, MA, USA), IL-6 antibody (1: 1000; Abcam, Cambridge, MA, USA), p53 antibody (1: 1000; Cell Signaling Technology, Inc., Danvers, MA, USA), β-actin antibody (1: 2000; Abcam, Cambridge, MA, USA), and secondary antibodies (1: 20 000; Merck Millipore, Darmstadt, Germany).
4
Western Blot Analysis of IL-6 and β-Actin
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Protein (50 μg) was separated by SDS‐ polyacrylamide gel electrophoresis and transferred onto PVDF (polyvinylidene fluoride) membrane (Bio-Rad Lab Inc., Hercules, CA) by electro blotting. The membrane was blocked with 5% nonfat dry milk in X1 TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.01% Tween 20) for 1 h at room temperature. The membrane was then incubated in primary antibody, IL-6 antibody (#ab6672, Abcam Inc, Cambridge, MA) or β-actin (#4970, Cell Signaling Technology Inc., Danvers, MA) at 1:1000 dilution in X1 TBST with 5% bovine serum albumin (Sigma Aldrich Inc., St. Louis, MO) overnight at 4 °C. The membrane was washed three times with X1 TBST for 10 min each and incubated with HRP‐conjugated secondary antibody (#7074, Cell Signaling Technology Inc., Danvers, MA) at 1:5000 dilution in X1 TBST with 5% nonfat dry milk for 1 h at room temperature. Following washes in X1 TBST, proteins were detected using the enhanced chemiluminescence system (Bio-Rad Lab Inc, Hercules, CA).
5
Neutralizing Adipocyte-Derived IL-6
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To differentiated human adipocytes in 6-well plate, 400 ug/ml IL-6 antibody (Abcam) was added to adipocytes media to neutralise IL-6 secreted by adipocytes.
6
Inflammation Factors in Gastrocnemius Muscle
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Expression of the proinflammation factors IL-1β and IL-6 in gastrocnemius muscle was detected by immunohistochemistry. The sections were deparaffinized with xylene and rehydrated with ethanol, and antigen retrieval was performed in 0.01 M citrate buffer (pH 6.0) in a pressure cooker, followed by natural cooling to room temperature. The sections were incubated in 0.3% hydrogen peroxide at room temperature for 10 min; goat serum was used to block the sections for 15 min at room temperature, which were then incubated overnight at 4°C with a rabbit polyclonal anti-IL-1 antibody (1:200 dilution) and IL-6 antibody (1:200 dilution) (both from Abcam, Cambridge, United Kingdom) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (ABclonal, Wuhan, China) for 30 min at room temperature. Immunodetection was performed using diaminobenzidine solution according to the manufacturer’s instructions. After washing, the sections were counterstained, dehydrated, and then coverslipped using neutral gum sealant.
7
Midbrain Neurodegeneration Model Protocol
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A frozen microtome (Leica, Germany), brain stereotaxic apparatus (Reward, China), fluorescence microscope (Carl Zeiss, Germany), electrophoresis apparatus and Transfer Machine (BioRad, USA), 10 μl micro-syringe (Stoelting, USA), P2X4R lentivirus (Genechem, China, Titer 2E + 9TU/ml), P2X4R antibody (Alamnelabs, Israel), IL-6 antibody (Abcam, UK), tyrosine hydroxylase (TH) antibody (Millipore, USA), goat-anti-rabbit IgG and goat-anti-mouse IgG (Life Technologies, USA), 6-hydroxydopamine (Sigma, USA), apomorphine (Sigma, USA), isoflurane, isopropanol, anhydrous ethanol (Fuyu Chemical, China).
8
Quantifying Hippocampal IL-6 Protein Levels
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IL-6 antibody (1:1,000 dilution; Abcam, Cambridge, MA, USA, Cat. Number: ab6672) was used to recognize IL-6 (24 kDa). Western blot quantification of the hippocampal tissues was performed as described by Xie et al. (2008). Briefly, signal intensity was analyzed using a Bio-Rad (Hercules, CA, USA) image program (Quantity One). We quantified Western blots in two steps. First, we used β-actin levels to normalize (e.g., determining ratio of IL-6 to β-actin) protein levels and control for loading differences in total protein amount. Second, we presented protein level changes in the mice receiving the surgery as a percentage of those in the sham group mice. 100% of protein level changes refer to control levels for the purpose of comparison to experimental conditions.
9
Emodin Regulates Neuroinflammation Pathways
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The following
materials and reagents were used in the study: Emodin (no.: A0044,
Chengdu Must Biotechnology Co., Ltd., China, purity≥98%); anti-MMP9
(no.: Ab38898, Abcam, U.K.); anti-NF-κB p65 (no.: #8242, CST,
USA); anti-IKKβ (no.: #2678, CST, USA); 2,3,5-triphenyl tetrazolium
chloride (no.: T8877-25G, Sigma, USA); hematoxylin and eosin (HE)
staining kit (no.:BP-DL017, SenBeiJia Biological Technology Co., Ltd.
China); HRP-labeled goat anti-rabbit IgG (H + L) (no.: A0208, Beyotime
Biotechnology, China); anti-MMP2 (no.: Ab181286), anti-occludin (no.:
Ab216327), anti-extracellular signal-regulated kinase [ERK1/2 (no.:
Ab184699)], and p-anti-ERK1/2 (no.: Ab214036)—Abcam, U.K.;
IL-6 antibody (no.: DF6087), HIF1A antibody (no./AF1009), claudin-5
(no./AF5216), and VEGFA antibody (no.: DF7470)—Affinity, China;
Alexa Fluor 555-labeled donkey anti-mouse IgG(H + L) (A0460), Triton
X-100 (no.: ST795), and antifade mounting medium (no.: P0126)—Beyotime
Biotechnology, China; neutral balsam (no.: G8590, Beijing Solarbio
Science & Technology Co., Ltd. China); RNA Easy Fast Tissue/Cell
Kit (no.: DP451), FastKing gDNA Dispelling RT SuperMix (no.: KR118),
and Talent qPCR PreMix (SYBR Green) (no.: FP209)—Tiangen Biotech
(Beijing) Co., Ltd, China; anti-GAPDH (no: AF7021, Affinity); and
BCA Protein Assay Kit (no: P0012S, Beyotime Biotechnology, China).
materials and reagents were used in the study: Emodin (no.: A0044,
Chengdu Must Biotechnology Co., Ltd., China, purity≥98%); anti-MMP9
(no.: Ab38898, Abcam, U.K.); anti-NF-κB p65 (no.: #8242, CST,
USA); anti-IKKβ (no.: #2678, CST, USA); 2,3,5-triphenyl tetrazolium
chloride (no.: T8877-25G, Sigma, USA); hematoxylin and eosin (HE)
staining kit (no.:BP-DL017, SenBeiJia Biological Technology Co., Ltd.
China); HRP-labeled goat anti-rabbit IgG (H + L) (no.: A0208, Beyotime
Biotechnology, China); anti-MMP2 (no.: Ab181286), anti-occludin (no.:
Ab216327), anti-extracellular signal-regulated kinase [ERK1/2 (no.:
Ab184699)], and p-anti-ERK1/2 (no.: Ab214036)—Abcam, U.K.;
IL-6 antibody (no.: DF6087), HIF1A antibody (no./AF1009), claudin-5
(no./AF5216), and VEGFA antibody (no.: DF7470)—Affinity, China;
Alexa Fluor 555-labeled donkey anti-mouse IgG(H + L) (A0460), Triton
X-100 (no.: ST795), and antifade mounting medium (no.: P0126)—Beyotime
Biotechnology, China; neutral balsam (no.: G8590, Beijing Solarbio
Science & Technology Co., Ltd. China); RNA Easy Fast Tissue/Cell
Kit (no.: DP451), FastKing gDNA Dispelling RT SuperMix (no.: KR118),
and Talent qPCR PreMix (SYBR Green) (no.: FP209)—Tiangen Biotech
(Beijing) Co., Ltd, China; anti-GAPDH (no: AF7021, Affinity); and
BCA Protein Assay Kit (no: P0012S, Beyotime Biotechnology, China).
10
Western Blot Quantification of IL-6 and PSD-95
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