Anti-VISTA 抗体 [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.

Blocking / Diluting buffer and concentration:

5% NFDM/TBST

Blocking / Diluting buffer and concentration:

5% NFDM/TBST

Blocking / Diluting buffer and concentration:

5% NFDM/TBST

Blocking / Diluting buffer and concentration:

5% NFDM/TBST

Blocking / Diluting buffer and concentration:

5% NFDM/TBST

Blocking / Diluting buffer and concentration:

5% NFDM/TBST

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. 

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.

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.

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

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