Anti-VISTA antibody [EPR21050]

• IHC-P

Supplier Data

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 ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Lab

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

• IHC-P

Supplier Data

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 ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Lab

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 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).

• IP

Supplier Data

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) (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 (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

false

• WB

Lab

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] (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

false

• WB

Supplier Data

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 34 kDa

false