Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with 50ng/ml IFN-γ for 16 hours or untreated labeling Indoleamine 2 3-dioxygenase with ab211017 at 1/2000 dilution followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

The signal increased after treatment with IFN-γ (50 ng/ml) for 16 hours on HeLa cells.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Tissue Microarrays stained for "Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374]" using "ab211017"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab211017 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Indoleamine 2 3-dioxygenase with ab211017 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Cytoplasmic and nuclear staining on dendritic cells of human tonsil is observed (PMID : 21328335 PMID : 25271151).

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling Indoleamine 2 3-dioxygenase with ab211017 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Cytoplasmic and nuclear staining on dendritic cells of human spleen is observed (PMID : 21328335 PMID : 25271151).

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Immunohistochemical analysis of paraffin-embedded human endometrium cancer tissue labeling Indoleamine 2 3-dioxygenase with ab211017 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Cytoplasmic and nuclear staining on human endometrium cancer is observed (PMID : 26155395).

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, treated with 50ng/ml IFN-γ for 16h (red) or untreated (green), labeling Indoleamine 2, 3-dioxygenase with ab211017 at 1/500 dilution compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling Indoleamine 2 3-dioxygenase with ab211017 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Cytoplasmic and nuclear staining on endothelial cells of human placenta is observed (PMID : 21328335 PMID : 25271151).

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Indoleamine 2, 3-dioxygenase was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate treated with 50ng/ml IFN-γ for 16h with ab211017 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab211017 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa treated with 50ng/ml IFN-γ for 16h whole cell lysate 10 μg (Input).

Lane 2 : ab211017 IP in HeLa treated with 50ng/ml IFN-γ for 16h whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab211017 in HeLa treated with 50ng/ml IFN-γ for 16h whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017)

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

• WB

Supplier Data

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1-3 : 3 minutes; Lane 4 : 15 seconds.

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017) at 1/1000 dilution

Lane 1:

Human ovary cancer lysate at 20 µg

Lane 2:

Human placenta lysate at 20 µg

Lane 3:

Human tonsil lysate at 20 µg

Lane 4:

SK-OV-3 (Human ovarian cancer cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

• WB

Lab

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Lanes 1 - 6 : Merged signal (red and green). Green - ab211017 observed at 40 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab211017 was shown to react with Indoleamine 2, 3-dioxygenase in treated wild-type A549 cells in Western blot with no signal observed in treated IDO1 knockout cell line ab266949 (IDO1 knockout cell lysate ab256948). Wild-type A549 and IDO1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab211017 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017) at 1/1000 dilution

Lane 1:

Wild-type A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate at 20 µg

Lane 2:

Wild-type A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg

Lane 2:

Western blot - Human IDO1 (Indoleamine 2, 3-dioxygenase) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-ido1-indoleamine-2-3-dioxygenase-knockout-a549-cell-line-ab266949'>ab266949</a>)

Lane 3:

IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/ml, 48 h) cell lysate at 20 µg

Lane 4:

IDO1 knockout A549 Treated IFN gamma (25 ng/ml, 48 h) cell lysate at 20 µg

Lane 5:

SK-OV-3 cell lysate at 20 µg

Lane 6:

MCF7 cell lysate at 20 µg

Predicted band size: 45 kDa

Observed band size: 40 kDa

false

• WB

Supplier Data

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (AB211017)

Blocking/Dilution buffer : 5% NFDM/TBST.

The level of Indoleamine 2, 3-dioxygenase expression can be induced by IFN-γ treatment (PMID 16368976).

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR20374] (ab211017) at 1/1000 dilution

Lane 1:

Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HeLa whole cell lysate treated with 50ng/ml Interferon-gamma (IFN-gamma, <a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-ab51240'>ab51240</a>) for 16 hours at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 45 kDa

Observed band size: 45 kDa

false

Exposure time: 15s