- 711778 - Invitrogen Antibodies IDO1 antibody

Submitted references Stromal-like Wilms tumor cells induce human Natural Killer cell degranulation and display immunomodulatory properties towards NK cells.

Cantoni C, Serra M, Parisi E, Azzarone B, Sementa AR, Nasto LA, Moretta L, Candiano G, Bottino C, Ghiggeri GM, Spaggiari GM
Oncoimmunology 2021 Mar 8;10(1):1879530

Recovery of Renal Function following Kidney-Specific VEGF Therapy in Experimental Renovascular Disease.

Engel JE, Williams ML, Williams E, Azar C, Taylor EB, Bidwell GL, Chade AR
American journal of nephrology 2020;51(11):891-902

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), HEK-293 treated with hIFN-γ (50 ng/mL, for 24hrs) (Lane 2), MDA-MB-231 (Lane 3), MDA-MB-231 treated with hIFN-γ (50 ng/mL, for 24hrs) (Lane 4), HT-1080 (Lane 5) and HT-1080 treated with hIFN-γ (50 ng/mL, for 24hrs) (Lane 6). The blots were probed with Anti-IDO1 Recombinant Rabbit Polyclonal Antibody (Product # 711778, 1 µg/mL). A 43 kDa band corresponding to IDO1, that specifically increases upon hIFN-γ treatment is as shown. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis, IFN gamma (50 ng/mL 24h) treated HeLa cells were fixed and permeabilized for detection of endogenous IDO1 using Anti-IDO1 Recombinant Rabbit Monoclonal Antibody (Product # 711778, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of IDO1 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localisation of IDO1. Panel e) shows no signal in untreated cells. Panel f) represents control cells with no primary antibody to assess background.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow Cytometry analysis of endogenous IDO1 in bone marrow derived mesenchymal stem cells was performed out using Anti-IDO Recombinant Rabbit Polyclonal Antibody (Product # 711778). Cells were either untreated or treated with 10 ng/mL IFN gamma for 40 hours, labeled with Anti-IDO1 Recombinant Rabbit Polyclonal Antibody (Product # 711778, 5 µg/ 1M cells) followed by detection with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 µg/mL, 1:2500). Rabbit IgG was used as the isotype control to determine nonspecific binding. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (Product # 4468770).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of IDO was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR864924_LV) and LentiArray Cas9 Lentivirus (Product # A32064). For Flow cytometry analysis, IDO Knock out cells were treated with 50 ng/mL IFN gamma for 24hrs and stained intracellularly using the intracellular Fixation & Permeabilization Buffer Set (Product # 88-8824-00) and protocol, with 2 µg/mL IDO Recombinant Polyclonal Antibody (Product # 711778, blue histogram) or with the 2 µg/mL Rabbit IgG Isotype Control (RbNP15), eBioscience™ (14-4616-82, yellow histogram) followed by Goat anti-Rabbit IgG (Heavy chain), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A55053, 1:1000). IFN gamma treated HeLa Cas9 control cells were also stained with the 2 µg/mL IDO Recombinant Polyclonal Antibody (Product # 711778, pink histogram) followed by the secondary antibody. Lossof signal was observed in the IDO KOcells stained with IDO antibody but not in the control Cas9cells. Viable cells were used for analysis, as determined by Fixable Viability Dye eFluor™780 (Product # 65-0865-18).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3. Representative immunostaining ( a ) and quantification ( b ) of M1 and M2 macrophages using the M1 marker IDO1 (red), the M2 marker MRC1 (green), and the nuclear counterstain DAPI (blue) in normal, RVD, and RVD + KTP-ELP-VEGF pigs after 10 weeks of observation. Untreated RVD pigs showed significantly increased macrophage infiltration which was markedly skewed towards inflammatory M1 polarization. Pigs treated with KTP-ELP-VEGF showed similar overall macrophage infiltration which was shifted in favor of anti-inflammatory M2s. Scale bar represents 20 mum; n = 6 per group. * p < 0.05 versus normal; + p < 0.05 versus RVD (single-way ANOVA and Tukey). IDO1, indoleamine-2,3-dioxygenase; MRC1, mannose receptor c-type 1; RVD, renovascular disease; KTP, kidney-targeting peptide; ELP, elastin-like polypeptide; VEGF, vascular endothelial growth factor.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5. Str-WT cells up-regulate inhibitory factors on exposure to inflammatory cytokines. (a) Western blot analysis of COX-2 expression in str-WT cells, and in g66 MSC, either unstimulated or stimulated with IL-1beta + TNF-alpha for 48 h. GAPDH expression was analyzed as positive control. Molecular weight (MW) markers (kDa) are indicated on the right. A representative experiment of three is shown. (b) Production of PGE2 by str-WT cells cultured alone (basal), stimulated by IL-1beta + TNF-alpha, or co-cultured with NK cells derived from two different healthy donors (NK1 and NK2). Culture supernatants were collected after 48 h (for cytokine stimulation experiments) or after 72 h (for co-culture experiments) and analyzed by ELISA assay. Data refer to the levels of PGE2 expressed as pg/ml. (c) Western blot analysis of IDO expression in str-WT cells and in g66 MSC, either unstimulated or stimulated with IFN-gamma for 48 h. GAPDH expression was analyzed as positive control. Molecular weight (MW) markers (kDa) are indicated on the right. A representative experiment of two is shown. (d) gW1 cells were stimulated with IFN-gamma and analyzed by flow cytometry for the expression of HLA-class I and the indicated immune checkpoint molecules. Light and dark gray histograms represent unstimulated and IFN-gamma-astimulated cells, respectively. Upper numbers represent MFI of unstimulated cells, while bold lower numbers refer to MFI of stimulated cells

711778

Antibody data