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Proteins and peptidesAnti-Ly6g antibody [1A8] - mouse IgG2c (Chimeric)
Low endotoxin, Azide free.
Our first-to-market chimera with mouse IgG2c backbone, this functional antibody specifically depletes neutrophils in vivo for up to 72h.
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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.
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
IHC-P | WB | Flow Cyt | IP | ICC/IF | |
---|---|---|---|---|---|
Human | Expected | Tested | Tested | Tested | Tested |
Mouse | Not recommended | Expected | Expected | Tested | Expected |
Rat | Not recommended | Expected | Expected | Expected | Expected |
Species | Dilution info | Notes |
---|---|---|
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. |
Species | Dilution info | Notes |
---|---|---|
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. |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/1000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes - |
Species Rat | Dilution info 1/1000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/500 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rat | Dilution info 1/500 | Notes - |
Species Mouse | Dilution info 1/500 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/30 | Notes - |
Species Human | Dilution info 1/30 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rat | Dilution info 1/30 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/50 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes - |
Species Rat | Dilution info 1/50 | Notes - |
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).
V-type immunoglobulin domain-containing suppressor of T-cell activation, Platelet receptor Gi24, Stress-induced secreted protein-1, V-set domain-containing immunoregulatory receptor, V-set immunoregulatory receptor, Sisp-1, PP2135, VSIR, UNQ730/PRO1412, VISTA, SISP1, C10orf54
Rabbit Monoclonal VISTA antibody. Suitable for IHC-P, WB, Flow Cyt, IP, ICC/IF and reacts with Human, Mouse, Rat samples.
V-type immunoglobulin domain-containing suppressor of T-cell activation, Platelet receptor Gi24, Stress-induced secreted protein-1, V-set domain-containing immunoregulatory receptor, V-set immunoregulatory receptor, Sisp-1, PP2135, VSIR, UNQ730/PRO1412, VISTA, SISP1, C10orf54
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
EPR25068-124
Affinity purification Protein A
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Avoid freeze / thaw cycle
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:
For more information, read more on recombinant antibodies.
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.
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.
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.
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.
We have tested this species and application combination and it works. It is covered by our product promise.
We have not tested this specific species and application combination in-house, but expect it will work. It is covered by our product promise.
This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our 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.
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
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
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
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
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
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
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
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
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
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
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
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 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.
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.
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.
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.
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.
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
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
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.
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.
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
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/20000 dilution
Observed band size: 50 kDa
Exposure time: 37s
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
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
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
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (AB97051) at 1/20000 dilution
Observed band size: 50 kDa, 70 kDa
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
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
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
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.
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.
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.
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
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 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.
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-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
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.
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.
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
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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