Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype control (Left) / HEK-293T (human embryonic kidney epithelial cell) transfected with an IDO1 expression vector containing a his tag (Middle) / HEK-293T transfected with IDO2 expression vector containing a his tag (Right) cells labelling Indoleamine 2, 3-dioxygenase with ab311847 at 1/500 dilution (0.1 ug)/Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human tissue labeling Indoleamine 2, 3-dioxygenase with ab311847 at 1/2000 (0.254 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on (A) HEK-293T transfected with an IDO1 expression vector containing a his tag. No staining on (B) HEK-293T transfected with IDO2 expression vector containing a his tag. The section was incubated with ab311847 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Indoleamine 2, 3-dioxygenase with ab311847 at 1/2000 (0.254 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on mouse liver (PMID : 19741271). The section was incubated with ab311847 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Immunohistochemical analysis of paraffin-embedded (A) C2C12 (mouse myo tissue labelling Indoleamine 2, 3-dioxygenase with ab311847 at 1/500 (1.016 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on (A) C2C12 treated with 50uM IFN-gamma for 24 hours cell pellet (treated). No staining on (B) C2C12 cell pellet (untreated). The section was incubated with ab311847 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype control (Left) /RAW 264.7 (mouse Abelson murine leukaemia virus-induced tumor macrophage) treated with 50ng/ml IFN-γ for 24h (Middle) / Untreated RAW 264.7 (Right) cells labelling Indoleamine 2, 3-dioxygenase with ab311847 at 1/500 dilution (0.1 ug)/Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Immunohistochemical analysis of paraffin-embedded Rat epididymis tissue labeling Indoleamine 2, 3-dioxygenase with ab311847 at 1/2000 (0.254 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat epididymis. The section was incubated with ab311847 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Immunohistochemical analysis of paraffin-embedded Mouse epididymis tissue labeling Indoleamine 2, 3-dioxygenase with ab311847 at 1/2000 (0.254 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse epididymis (PMID : 19741271). The section was incubated with ab311847 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat-mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Immunohistochemical analysis of paraffin-embedded Mouse small intestine tissue labelling Indoleamine 2, 3-dioxygenase with ab311847 at 1/2000 (0.254 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse small intestinal secretory epithelial cells (PMID : 31325428). The section was incubated with ab311847 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Indoleamine 2, 3-dioxygenase with ab311847 at 1/2000 (0.254 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on rat liver. The section was incubated with ab311847 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype control (Left) / C2C12 (mouse myoblast) treated with 50ng/ml IFN-γ for 24h (Middle) / Untreated C2C12 (Right) cells labelling Indoleamine 2, 3-dioxygenase with ab311847 at 1/500 dilution (0.1 ug)/Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Immunohistochemical analysis of paraffin-embedded (A) Colon tissue from wild-type C57BL/6J mice and (B) Colon tissue from Indoleamine 2, 3-dioxygenase knockout mice tissue labeling Indoleamine 2, 3-dioxygenase with ab311847 at 1/2000 dilution.

Positive staining on (A) Colon tissue from wild-type C57BL/6J mice, no staining on (B) Colon tissue from Indoleamine 2, 3-dioxygenase knockout mice.

The primary antibody was incubated for 30 mins at room temperature, incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Ido1-KO homozygous mice (Strain ID : T011654).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Immunohistochemical analysis of paraffin-embedded (A) Ileum tissue from wild-type C57BL/6J mice and (B) Ileum tissue from Indoleamine 2, 3-dioxygenase knockout mice tissue labeling Indoleamine 2, 3-dioxygenase with ab311847 at 1/2000 dilution, followed by a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Positive staining on (A) Ileum tissue from wild-type C57BL/6J mice, no staining on (B) Ileum tissue from Indoleamine 2, 3-dioxygenase knockout mice.

The primary antibody was incubated for 30 mins at room temperature, incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond^™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Ido1-KO homozygous mice (Strain ID : T011654).

• IP

Supplier Data

Immunoprecipitation - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Indoleamine 2, 3-dioxygenase was immunoprecipitated from 0.35 mg C2C12 (mouse myoblast) treated with 50 ng/ml IFN gamma for 24 h, whole cell lysate with ab311847 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab311847 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : C2C12 (mouse myoblast) treated with 50 ng/ml IFN gamma for 24 h, whole cell lysate Lane 2 : C2C12 (mouse myoblast) treated with 50 ng/ml IFN gamma for 24 h, whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab311847 in C2C12 treated with 50 ng/ml IFN gamma for 24 h whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (ab311847) at 1/30 dilution

All lanes:

C2C12 (mouse myoblast) treated with 50 ng/ml IFN gamma for 24 h, whole cell lysate at 20 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 45 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Western blot : Anti-IDO1 antibody [EPR28349-89] (ab311847) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab311847 was shown to bind specifically to IDO1. A band was observed at 42 kDa in treated wild-type A549 cell lysates with no signal observed at this size in IDO1 knockout cell line. To generate this image, wild-type and IDO1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (ab311847) at 1/1000 dilution

Lane 1:

Wild-type A549 Treated IFN gamma (25 ng/mL, 48 h) ab281500 cell lysate at 20 µg

Lane 2:

Wild-type A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab255554 cell lysate at 20 µg

Lane 3:

IDO1 knockout A549 Treated IFN gamma (25 ng/mL, 48 h) ab281489 cell lysate at 20 µg

Lane 4:

IDO1 knockout A549 Vehicle Control IFN gamma (0 ng/mL, 48 h) ab263515 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 42 kDa

false

• WB

Supplier Data

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression of Indoleamine 2, 3-dioxygenase is upregulated in response to IFN gamma treatment (PMID : 30809284, 34735466). In Western blot, anti-H3 antibody (ab176842) loading control staining at 1/100000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (ab311847) at 1/1000 dilution

Lane 1:

Untreated C2C12 (mouse myoblast) whole cell lysate at 20 µg

Lane 2:

C2C12 treated with /ml IFN gamma for 24 h, whole cell lysate at 20 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

Observed band size: 45 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression of Indoleamine 2, 3-dioxygenase is upregulated in response to IFN gamma treatment (PMID : 30809284, 34735466). In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (ab311847) at 1/1000 dilution

Lane 1:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW264.7 treated with /ml IFN gamma for 24 h, whole cell lysate at 20 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

Observed band size: 45 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (AB311847)

Exposure time : Lane 1-4 : 1 second; Lane 5-8 : 6 seconds; Lane 9-12 : 15 seconds.

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

In Western blot, Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002) staining at 1/5000 dilution.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Ido1-KO homozygous mice (Strain ID : T011654).

All lanes:

Western blot - Anti-Indoleamine 2, 3-dioxygenase antibody [EPR28349-89] (ab311847) at 1/1000 dilution

Lane 1:

Wild-type mouse epididymis tissue lysate (male case1) at 40 µg

Lane 2:

Wild-type mouse epididymis tissue lysate (male case2) at 40 µg

Lane 3:

Ido1 knockout mouse epididymis tissue lysate (male case1) at 40 µg

Lane 4:

Ido1 knockout mouse epididymis tissue lysate (male case2) at 40 µg

Lane 5:

Wild-type mouse ileum tissue lysate (female case) at 40 µg

Lane 6:

Wild-type mouse ileum tissue lysate (male case) at 40 µg

Lane 7:

Ido1 knockout mouse ileum tissue lysate (male case1) at 40 µg

Lane 8:

Ido1 knockout mouse ileum tissue lysate (male case2) at 40 µg

Lane 9:

Wild-type mouse colon tissue lysate (female case) at 40 µg

Lane 10:

Wild-type mouse colon tissue lysate (male case) at 40 µg

Lane 11:

Ido1 knockout mouse colon tissue lysate (male case1) at 40 µg

Lane 12:

Ido1 knockout mouse colon tissue lysate (male case2) at 40 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 1/20000 dilution

Observed band size: 45 kDa

false