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Rabbit Monoclonal VISTA antibody. Suitable for IHC-P, WB, Flow Cyt, IP, ICC/IF and reacts with Human, Mouse, Rat samples.

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Images

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.
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Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
IHC-PWBFlow CytIPICC/IF
Human
Expected
Tested
Tested
Tested
Tested
Mouse
Not recommended
Expected
Expected
Tested
Expected
Rat
Not recommended
Expected
Expected
Expected
Expected

Expected
Expected

Species

Human

Dilution info

1/100

Notes

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

Not recommended
Not recommended

Species

Rat

Dilution info

-

Notes

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

Species

Mouse

Dilution info

-

Notes

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

Tested
Tested

Species

Human

Dilution info

1/1000

Notes

-

Expected
Expected

Species

Mouse

Dilution info

-

Notes

-

Species

Rat

Dilution info

1/1000

Notes

-

Tested
Tested

Species

Human

Dilution info

1/500

Notes

-

Expected
Expected

Species

Rat

Dilution info

1/500

Notes

-

Species

Mouse

Dilution info

1/500

Notes

-

Tested
Tested

Species

Mouse

Dilution info

1/30

Notes

-

Species

Human

Dilution info

1/30

Notes

-

Expected
Expected

Species

Rat

Dilution info

1/30

Notes

-

Tested
Tested

Species

Human

Dilution info

1/50

Notes

-

Expected
Expected

Species

Mouse

Dilution info

-

Notes

-

Species

Rat

Dilution info

1/50

Notes

-

Target data

Function

Immunoregulatory receptor which inhibits the T-cell response (PubMed:24691993). May promote differentiation of embryonic stem cells, by inhibiting BMP4 signaling (By similarity). May stimulate MMP14-mediated MMP2 activation (PubMed:20666777).

Alternative names

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Rabbit Monoclonal VISTA antibody. Suitable for IHC-P, WB, Flow Cyt, IP, ICC/IF and reacts with Human, Mouse, Rat samples.

Alternative names

Key facts

Isotype

IgG

Form

Liquid

Clonality

Monoclonal

Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number

EPR25068-124

Purification technique

Affinity purification Protein A

Concentration
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Storage

Shipped at conditions

Blue Ice

Appropriate short-term storage duration

1-2 weeks

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

-20°C

Aliquoting information

Upon delivery aliquot

Storage information

Avoid freeze / thaw cycle

Notes

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

  • - High batch-to-batch consistency and reproducibility
  • - Improved sensitivity and specificity
  • - Long-term security of supply
  • - Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

Biological function summary

VISTA functions in immune checkpoint regulation by modulating T cell activity. It acts as both a ligand and a receptor influencing immune responses. As part of an immune complex VISTA interacts with other checkpoint proteins to maintain immune homeostasis and prevent autoimmunity. Its role mirrors those of other B7 family members like PD-1 and CTLA-4 which are involved in negative immune regulation.

Activity summary

VISTA also known as V-domain Ig suppressor of T cell activation or Gi24 is a protein significant in immune regulation. It is a type I transmembrane glycoprotein with an apparent mass of approximately 55 kDa. VISTA is expressed on hematopoietic cells specifically within immune system components such as myeloid cells and T cells. Researchers have identified biotinylated chimeric proteins which facilitate the study of VISTA's expression and interactions in various immunological research models.

Pathways

VISTA integrates into the broader framework of immune signaling pathways. It contributes significantly to the immune checkpoint pathway similar to CTLA-4 and PD-1 which mediate immune cell proliferation and activation. By interacting with these proteins VISTA plays an important role in maintaining self-tolerance and regulating inflammatory responses functioning parallel to other proteins such as CD28 and B7-H5 in the immune checkpoint pathway.

Associated diseases and disorders

VISTA's improper regulation associates with autoimmune diseases and cancer. Its expression influences the progression of conditions like rheumatoid arthritis where it modulates immune responses and inflammation. Additionally in oncology VISTA acts as an inhibitory molecule; hence targeting it can enhance anti-tumor immunity similar to therapies targeting proteins like PD-1 and PD-L1. Understanding VISTA's intricate connections helps in developing therapies for these diseases.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

30 product images

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    Lane 1: bEnd.3 (mouse brain endothelioma) whole cell lysate treated with Protein Deglycosylation MIX II at 15 µg

    Lane 2: Untreated bEnd.3 whole cell lysate at 15 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051)

    Predicted band size: 34 kDa

    Observed band size: 34 kDa, 50 kDa

    Exposure time: 125s

    This data was developed using ab300042, the same antibody clone in a different buffer formulation.

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    Lane 1: bEnd.3 (mouse brain endothelioma) whole cell lysate at 20 µg

    Lane 2: EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

    Lane 3: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 50 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/20000 dilution

    Predicted band size: 34 kDa

    Observed band size: 50 kDa, 70 kDa

    This data was developed using ab300042, the same antibody clone in a different buffer formulation.

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    Lane 1: DU-145 (Human prostate carcinoma epithelial) whole cell lysate treated with Protein Deglycosylation MIX II at 15 µg

    Lane 2: Untreated DU-145 whole cell lysate at 15 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/50000 dilution

    Predicted band size: 34 kDa

    Observed band size: 34 kDa, 50 kDa

    Exposure time: 48s

    This data was developed using ab300042, the same antibody clone in a different buffer formulation.

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    All lanes: Human heart tissue lysate at 20 µg

    Secondary

    All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

    Predicted band size: 34 kDa

    Observed band size: 50 kDa

    Exposure time: 59s

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    Lane 1: Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

    Lane 2: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

    Lane 3: HEK-293T (human embryonic kidney) whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/20000 dilution

    Predicted band size: 34 kDa

    Observed band size: 50 kDa

    Exposure time: 37s

    This data was developed using ab300042, the same antibody clone in a different buffer formulation.

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration:

    5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    All lanes: Human spleen tissue lysate at 20 µg

    Secondary

    All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

    Predicted band size: 34 kDa

    Observed band size: 37-50 kDa

    Exposure time: 10s

  • Flow Cytometry - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Flow Cytometry - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Flow cytometric analysis of Human peripheral blood mononuclear cells (PBMC) labeling VISTA with ab300042 at 1/500 dilution (0.1 ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Cells were stained with rabbit IgG or ab300042. Then stained with anti-CD14 conjugated to Alexa Fluor® 647. Gated on viable cells.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling VISTA with ab300042 at 1/100 dilution (4.92 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human tonsil. The section was incubated with ab300042 for 30 mins at room temperature. 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling VISTA with ab300042 at 1/100 dilution (4.92 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human placenta. The section was incubated with ab300042 for 30 mins at room temperature.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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling VISTA with ab300042 at 1/100 dilution (4.92 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on endothelial cells of human cerebrum. The section was incubated with ab300042 for 30 mins at room temperature. 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human non-Hodgkin's B-Cell lymphoma tissue labeling VISTA with ab300042 at 1/100 dilution (4.92 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) was used. Positive staining on human non-Hodgkin's B-Cell lymphoma. The section was incubated with ab300042 for 30 mins at room temperature. 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

  • Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (ab300042)

    VISTA was immunoprecipitated from 0.35 mg DU-145 (Human prostate carcinoma epithelial) whole cell lysate with ab300042 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300042 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution.

    Lane 1: DU-145 (Human prostate carcinoma epithelial) whole cell lysate 10 μg

    Lane 2: ab300042 in DU-145 whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300042 in DU-145 whole cell lysate

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

    Exposure time: 32.368 seconds

    All lanes: Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (AB300042)

    Predicted band size: 34 kDa

  • Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (ab300042)

    VISTA was immunoprecipitated from 0.35 mg bEnd.3 (mouse brain endothelioma) whole cell lysate with ab300042 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300042 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution.

    Lane 1: bEnd.3 (mouse brain endothelioma) whole cell lysate 10 μg

    Lane 2: ab300042 in bEnd.3 whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300042 in bEnd.3 whole cell lysate

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

    Exposure time: 32 seconds

    All lanes: Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (AB300042)

    Predicted band size: 34 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling VISTA with ab300042 at 1/100 dilution (4.92 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on human spleen. The section was incubated with ab300042 for 30 mins at room temperature. 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

  • Immunocytochemistry/ Immunofluorescence - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Human PBMC cells lebeling VISTA with ab300042 at 1/50 (9.84 μg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 μ/ml) dilution (Green). Confocal image showing cytoplasmic and membranous staining in subsets of human PBMCs. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 μg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

    Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 μg/ml) dilution.

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration: 5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    Lane 1: Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

    Lane 2: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

    Lane 3: HEK-293T (Human embryonic kidney) whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/20000 dilution

    Observed band size: 50 kDa

    Exposure time: 37s

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration: 5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    All lanes: Human heart tissue lysate at 20 µg

    Secondary

    All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

    Observed band size: 50 kDa

    Exposure time: 59s

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration: 5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    Lane 1: bEnd.3 (Mouse brain endothelioma) whole cell lysate at 20 µg

    Lane 2: EL4 (Mouse lymphoma T lymphocyte) whole cell lysate at 20 µg

    Lane 3: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 50 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/20000 dilution

    Observed band size: 50 kDa, 70 kDa

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration: 5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    Lane 1: DU-145 (Human prostate carcinoma epithelial) whole cell lysate treated with Protein Deglycosylation MIX II at 15 µg

    Lane 2: Untreated DU-145 whole cell lysate at 15 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/50000 dilution

    Observed band size: 34 kDa, 50 kDa

    Exposure time: 48s

  • Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (ab300042)

    VISTA was immunoprecipitated from 0.35 mg bEnd.3 (mouse brain endothelioma) whole cell lysate with ab300042 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300042 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution. Lane 1: bEnd.3 (mouse brain endothelioma) whole cell lysate 10 µg. Lane 2: ab300042 in bEnd.3 whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300042 in bEnd.3 whole cell lysate. Blocking and dilution buffer and concentration: 5% NFDM/TBST. Exposure time: 32 seconds

    All lanes: Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/30 dilution

    Lane 1: bEnd.3 (Mouse brain endothelioma) at 10 µg

    Lane 2: ab300042 in bEnd.3 whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300042 in bEnd.3 whole cell lysate

  • Immunocytochemistry - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunocytochemistry - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Human PBMC cells lebeling VISTA with ab300042 at 1/50 (9.84 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 µ/ml) dilution (Green). Confocal image showing cytoplasmic and membranous staining in subsets of human PBMCs. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 µg/ml) dilution.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling VISTA with ab300042 at 1/100 dilution (4.92 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human spleen. The section was incubated with ab300042 for 30 mins at room temperature. 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling VISTA with ab300042 at 1/100 dilution (4.92 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human placenta. The section was incubated with ab300042 for 30 mins at room temperature. 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Blocking / Diluting buffer and concentration: 5% NFDM/TBST

    All lanes: Western blot - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    All lanes: Human spleen tissue lysate at 20 µg

    Secondary

    All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/1000 dilution

    Observed band size: 37,50 kDa

    Exposure time: 10s

  • Flow Cytometry - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Flow Cytometry - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Flow cytometric analysis of Human peripheral blood mononuclear cells (PBMC) labeling VISTA with ab300042 at 1/500 dilution (0.1 ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Cells were stained with rabbit IgG or ab300042. Then stained with anti-CD14 conjugated to Alexa Fluor® 647. Gated on viable cells.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling VISTA with ab300042 at 1/100 dilution (4.92 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on endothelial cells of human cerebrum. The section was incubated with ab300042 for 30 mins at room temperature. 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

  • Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Western blot - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Western blot: Anti-VSIR antibody [EPR25068-124] (ab300042) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab300042 was shown to bind specifically to VSIR. A band was observed at 50-60 kDa in wild-type A549 cell lysates with no signal observed at this size in VSIR knockout cell line. To generate this image, wild-type and VSIR 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-VISTA antibody [EPR25068-124] (AB300042) at 1/1000 dilution

    Lane 1: Wild-type A549 cell lysate at 20 µg

    Lane 2: VSIR knockout A549 cell lysate at 20 µg

    Lane 3: DU 145 cell lysate at 20 µg

    Lane 4: MCF7 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

    Performed under reducing conditions.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling VISTA with abab300042 at 1/100 dilution (4.92 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human tonsil. The section was incubated with ab300042 for 30 mins at room temperature. 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

  • Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (ab300042)

    VISTA was immunoprecipitated from 0.35 mg DU-145 (Human prostate carcinoma epithelial) whole cell lysate with ab300042 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300042 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution. Lane 1: DU-145 (Human prostate carcinoma epithelial) whole cell lysate 10 µg. Lane 2: ab300042 in DU-145 whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300042 in DU-145 whole cell lysate. Blocking and dilution buffer and concentration: 5% NFDM/TBST. Exposure time: 32.368 seconds

    All lanes: Immunoprecipitation - Anti-VISTA antibody [EPR25068-124] (AB300042) at 1/30 dilution

    Lane 1: DU-145 (Human prostate carcinoma epithelial) whole cell lysate at 10 µg

    Lane 2: ab300042 in DU-145 whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300042 in DU-145 whole cell lysate

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR25068-124] (ab300042)

    Immunohistochemical analysis of paraffin-embedded Human non-Hodgkin's B-Cell lymphoma tissue labeling VISTA with ab300042 at 1/100 dilution (4.92 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on human non-Hodgkin's B-Cell lymphoma. The section was incubated with ab300042 for 30 mins at room temperature. 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

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