Anti-VISTA antibody [EPR21050]

7 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR21050] (ab230950)

  Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling VISTA with ab230950 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Membranous and cytoplasmic staining in tumor-infiltrating leukocytes of human lung cancer (PMID: 28236118, PMID: 28507801) is observed. Counterstained with hematoxylin.
  
  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
  
  Perform heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).

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  Immunoprecipitation - Anti-VISTA antibody [EPR21050] (ab230950)

  VISTA was immunoprecipitated from 0.35 mg DU145 (Human prostate carcinoma epithelial cell) whole cell lysate with ab230950 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab230950 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/5000 dilution.
  Lane 1: DU154 whole cell lysate 10 μg (Input).
  Lane 2: ab230950 IP in DU154 whole cell lysate.
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab230950 in DU154 whole cell lysate.
  Blocking/Dilution buffer: 5% NFDM/TBST.
  Exposure time: 3 minutes.

  All lanes: Immunoprecipitation - Anti-VISTA antibody [EPR21050] (ab230950)

  Predicted band size: 34 kDa

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  Western blot - Anti-VISTA antibody [EPR21050] (ab230950)

  Blocking/Dilution buffer: 5% NFDM/TBST.
  

  Exposure times: Lanes 1-2: 32 seconds; Lanes 3-4: 6 seconds.

  The 50-60 kda band observed is the glycosylated form of VISTA, consistent with what has been described in the literature (PMID: 29216931).

  All lanes: Western blot - Anti-VISTA antibody [EPR21050] (ab230950) at 1/1000 dilution

  Lane 1: Human fetal spleen lysate at 20 µg

  Lane 2: Human thymus lysate at 20 µg

  Lane 3: DU145 (Human prostate carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 4: Human fetal lung lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 34 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR21050] (ab230950)

  Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling VISTA with ab230950 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Membranous and cytoplasmic staining in leukocytes of human spleen (PMID: 21383057, PMID: 24691993) is observed. Counterstained with hematoxylin.
  
  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
  
  Perform heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).

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  Western blot - Anti-VISTA antibody [EPR21050] (ab230950)

  Western blot: Anti-VSIR antibody [EPR21050] (ab230950) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab230950 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 [EPR21050] (ab230950) 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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR21050] (ab230950)

  Immunohistochemical analysis of formalin fixed paraffin (FFPE) embedded tonsil labelling VISTA with ab230950 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 64 min with DISCOVERY cell conditioning solution (CC1)  100°C, pH 8.5. ab230950 was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VISTA antibody [EPR21050] (ab230950)

  Tissue Microarrays stained for "Anti-VISTA antibody [EPR21050]" using "ab230950"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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab230950 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).