Human Huh7.5.1 cells were kindly provided by Dr. F. Chisari (The Scripps Research Institute, La Jolla, CA; MTA number 636) [18 (link)]. PHH were provided by Stephen Strom of University of Pittsburgh through the Liver Tissue Procurement and Distribution System (N01-DK-7-0004/HHSN26700700004C). PHH were isolated from liver resections according to standard perfusion protocols. After seven days of culture, PHH remained attached and maintained a healthy and highly differentiated phenotype. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors (EFS-Alsace, Strasbourg, France) by Ficoll density gradient centrifugation. CD4+ T cell positive selection was performed by magnetic sorting using CD4+ microbeads (Miltenyi Biotech). Human recombinant IFN-α and IFN-γ were obtained from Roche and Intermune, respectively. Rabbit polyclonal anti-IDO1 (Abcam), anti-β actin (Abcam), anti-IRF1 (Santa Cruz Biotechnology), anti-STAT1 and anti-STAT1 (Tyr701) antibody (Cell Signaling) were used for Western blot analysis. PE-conjugated mouse anti-human IFN-γR, anti-HCV core (clone C7-50, Thermo Scientific) and anti-IRF1 antibodies were used for flow cytometry analysis. The IDO inhibitor 1-DL-MT, a racemic mixture of 1-methyl-tryptophan comprising both isomers (D+L), was purchased from Sigma-Aldrich.
Anti ido1
Manufactured by Abcam
Sourced in United Kingdom
Anti-IDO1 is a laboratory reagent that targets the protein indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 is an enzyme involved in the metabolism of the amino acid tryptophan. This reagent can be used in research applications to study the role of IDO1 in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Abcam (3 mentions)
Anti tdo 2, by Abcam (2 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Bioworld Technology (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Anti irf1, by Santa Cruz Biotechnology (1 mentions)
Cd4 microbeads, by Miltenyi Biotec (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Anti ox camkii, by Merck Group (1 mentions)
Goat anti mouse igg horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting detection system, by GE Healthcare (1 mentions)
Goat anti rabbit, by Santa Cruz Biotechnology (1 mentions)
Chemidoc xrs machine, by Bio-Rad (1 mentions)
Anti fas, by Proteintech (1 mentions)
Image j analysis software, by Bio-Rad (1 mentions)
Bradford assay, by Beyotime (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
11 protocols using anti ido1
1
Isolation and Culture of Hepatic and Immune Cell Populations
2
Immunohistochemical Analysis of IDO-1
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Rabbit polyclonal anti-IDO-1 (Abcam, Cambridge, MA) was used at 1/50 dilution for overnight refrigerated incubation. Human tonsil was used as control. In lymphoid tissues, IDO is expressed in mature dendritic cells with a phenotype distinct from plasmacytoid dendritic cells (Fig. 1 ) [32 (link)].
3
Western Blot Analysis of IDO1 and CaMKII
4
Protein Analysis using Western Blotting
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Total protein lysates were obtained by lysing the cells with RIPA buffer supplemented with a phosphatase inhibitor cocktail (BioTeke, China). The protein content was measured by using the Bradford assay (Beyotime, China). Then, 20–40 μg of each sample was mixed with loading buffer and boiled for 5 min. All the samples were electrophoresed in a polyacrylamide gel and electro-blotted onto a polyvinylidene fluoride (PVDF) membrane. All the primary antibodies were used in accordance with to the manufacturer's instructions (anti-GAPDH, Proteintech, 10,494–1-AP; anti-SNF5, Proteintech, 20,654–1-AP; anti-BAF60c, Proteintech, 12,838–1-AP; anti-vimentin, Santa, sc-373,717; anti-E-cadherin, CST, #3195; anti-N-cadherin, CST, #13,116 anti-PD-L1, CST, #13,684; anti-Phospho-Stat3 (Tyr705), CST, #9131; anti-Stat3, CST, #9139; anti-PD-L2, ABCAM, ab187662; anti- IDO1, ABCAM, ab211017; anti- FAS, Proteintech, 13,098–1-AP; anti- SNAI1, Zen-bio, 340,942; anti-ZEB1, Zen-bio, 220,860; anti-β-Actin, Proteintech, 20,536–1-AP). The western blots were visualized under chemiluminescence conditions (Millipore, USA) using the ChemiDoc XRS machine (Bio-rad, USA). The bands were analyzed using the Image J Analysis software (Bio-rad, USA).
5
Immunohistochemical Analysis of Tumor Markers
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Paraffin-embedded tumor tissue sections were cut. After dewaxing and rehydration, sections were treated with antigen retrieval solution for 15 min at 100 °C. Subsequently, samples were blocked with 5 % BSA in PBST for 30 min. Then slides were incubated with anti-TDO2 (1:200, Abcam, UK), anti-IDO1 (1:200, Abcam, UK), anti-IL-6 (1:200, Abcam, UK), anti-Kyn (1:200, Abcam, UK), anti-pSTAT3 (1:200, Abcam, UK), anti-NF-kB (1:200, Abcam, UK) antibodies overnight at 4 °C. After that, samples were incubated with HRP-conjugated secondary antibody (1:1000, Thermo, USA), developed by 3,3-diaminobenzidine solution and counterstained with hematoxylin. The intensity of protein expression in immunohistochemistry was determined by Image-Pro Plus 6.0 software. 8 tumor tissues from 8 patients were included in each group. 10 fields in each tumor tissue were analyzed and the mean of 10 values was determined as the expression intensity of this tumor tissues.
6
Western Blot Analysis of IDO1, CXCL10, and NF-κB
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EC cells protein were blotted as described in the literature [35 (link)]. In brief, protein was separated by electrophoresis on SDS-polyacrylamide electrophoresis (PAGE) gels, transferred to polyvinylidene difluoride (PVDF) membranes and blocked with blocking solution at room temperature for 1 h. The PVDF membranes were incubated with primary antibody (anti-IDO1, 1:750, Abcam, Cambridge, UK; anti-CXCL10, 1:1000, Saier, Tianjin, China; anti-P65, 1:2000, Proteintech, Wuhan, China; anti-p-P65, 1:1000, Abcam; P105, 1:1000, Abcam; anti-p-P105, 1:1000, Abcam; anti-GAPDH, 1:3000, Bioworld) at 4 °C overnight, then cleaned with 1×PBST buffer and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG/Goat Anti-Mouse, 1:3000, Beyotime, Shanghai, China) for 1 h at room temperature. After the membranes were washed in PBST, the blots were visualized by enhanced chemiluminescence ECL kit (Thermo Fisher Scientific).
7
Immunohistochemical Evaluation of IDO1 Expression in Esophageal Cancer Tissues
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Tissue microarrays consisting of EC tissue (n = 61) and normal esophageal tissue (n = 88) were fabricated according to the procedures provided in the literature [34 (link)]. Briefly, tissue slides were dewaxed with xylene, washed with alcohol, repaired with citrate, and blocked with hydrogen peroxide and 5% serum. The tissues were incubated with primary antibody (anti-IDO1, 1:200, Abcam, Cambridge, UK) at 4 °C overnight, then cleaned with 1×PBST buffer and incubated with secondary antibody (goat anti-rabbit IgG, 1:400, Beyotime, Shanghai, China) for 1 h at room temperature. Tissue slides were stained with DAB and hematoxylin to avoid light and then sealed with neutral gum. Finally, two pathologists scored independently according to the rate of positive cells and the intensity of staining color. Scores above the median were defined as high expression, otherwise low expression.
8
Immunosuppressive Pathway Profiling in Tumor Cells
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Tumor cells were collected and lysed in RIPA buffer (Byotime, China) with protease and phosphatase Inhibitor Cocktails (Biyuntian, China). Aliquots of 10 µg protein samples were separated by electrophoresis on 10 % SDS-polyacrylamide gels and transferred to a methanol‐pretreated PVDF membrane (Thermo, USA). Then samples were blocked with 5 % BSA in TBST and incubated with anti‐TDO2 (1:1000, Abcam, UK), anti-IDO1 (1:1000, Abcam, UK), anti‐AhR (1:1000, Abcam, UK), anti-STAT3 (1:1000, Abcam, UK), anti-TIM4 (1:1000, Abcam, UK) and anti‐β‐actin (1:1000, Abcam, UK) primary antibodies. Samples were visualized with an HRP‐conjugated secondary antibody (1:1000, Thermo, USA) and western blotting analysis system (Tanon, China).
9
Western Blot Analysis of Apoptosis Markers
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Cells were pelleted and lysed using lysis buffer (Roche). The protein concentration was determined. The samples were migrated on SDS-PAGE and transferred onto PVDF membranes (Roche). After blocking with 5% nonfat milk, the membrane was incubated with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Signals were detected using an enhanced chemiluminescence reagent (Millipore) and subjected to the Alpha Innotech Fluor Chem-FC2 imaging system (Alpha Innotech, San Leandro, CA). Antibodies were as follows: anti-GAPDH (Bioworld, Nanjing, China, 1:5000 diluted), anti-Caspase-3 (Cell Signaling Technology, 1:1000 diluted), anti-IDO-1 (Abcam, 1:500 diluted).
10
FFPE Lung Cancer Immunohistochemistry
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A total of 78 formalin-fixed, paraffin-embedded (FFPE) tissues (68 lung adenocarcinoma tissue specimens and 10 adjacent lung tissues) were obtained from 68 patients for immunohistochemical staining following routine protocol. Briefly, the FFPE tissues were sliced into sections at a thickness of 4 μm and roasted at 75°C followed by blocking with 2% normal goat serum. The specimens were then incubated with the following rabbit or mouse monoclonal primary antibodies at 4°C overnight: anti-IDO1, anti-CD3, anti-CD4, and anti-CD8 (all antibodies were purchased from Abcam, Shanghai, China). After washing, HRP-conjugated anti-rabbit or anti-mouse antibodies were applied for 1 h at room temperature. Staining was visualized with DAB substrate followed by counterstaining with hematoxylin.
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