Anti-VCAM1 antibody [EPR5047] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (AB271899)

Immunohistochemical analysis of formalin-fixed paraffin-embedded human tonsil labelling VCAM1 with ab271899 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 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab271899 anti VCAM1 antibody 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (AB271899)

Immunohistochemical staining of paraffin embedded human tonsil with purified ab134047 at a dilution of 1/500. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134047).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (AB271899)

Intracellular Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labeling VCAM1 with purified ab134047 at 1/40 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134047).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (AB271899)

Immunofluorescent staining of K562 cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab134047 at a dilution of 1/250. An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/500 and the cells were counter stained with DAPI. The negative control is shown in the bottom right hand panel - for the negative control, Alex Fluor^® 594 goat anti-mouse was used at a dilution of 1/500.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134047).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (AB271899)

ab134047 staining VCAM1 in HUVEC cells treated with TNF-α (ab55237) at 10 ng/ml for 16 hours. The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab134047 at 2μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 647) (ab150119) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134047).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (AB271899)

IHC image of VCAM1 staining in Human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab134047, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134047).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (AB271899)

IHC image of VCAM1 staining in Mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab134047, 1/200 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134047).

• IP

Supplier Data

Immunoprecipitation - Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (AB271899)

VCAM1 was immunoprecipitated from Human fetal liver lysate with ab134047 at 1/110 dilution.

Western blot was performed from the immunoprecipitate using ab134047 at 1/1000 dilution.

VeriBlot for IP secondary antibody (Peroxidase conjugated),was used as secondary antibody at 1/1000 dilution.

Lane 1 : Human fetal liver lysate

Blocking and dilution buffer : 5% NFDM/TBST

Exposure time : 1 second

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134047).

All lanes:

Immunoprecipitation - Anti-VCAM1 antibody [EPR5047] (<a href='/en-us/products/primary-antibodies/vcam1-antibody-epr5047-ab134047'>ab134047</a>)

Predicted band size: 81 kDa

false

• WB

Lab

Western blot - Anti-VCAM1 antibody [EPR5047] - BSA and Azide free (AB271899)

This data was developed using the same antibody clone in a different buffer formulation (ab134047).

Lanes 1 - 6 : Merged signal (red and green). Green - ab134047 observed at 105 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab134047 was shown to react with VCAM1 in wild-type A549 cells in Western blot with loss of signal observed in VCAM1 knockout sample. Wild-type A549 and VCAM1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab134047 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-VCAM1 antibody [EPR5047] (<a href='/en-us/products/primary-antibodies/vcam1-antibody-epr5047-ab134047'>ab134047</a>) at 1/2000 dilution

Lane 1:

Wild-type A549 cell lysate at 30 µg

Lane 2:

Wild-type A549 TNF-a treated (10 ng/mL, 16h) cell lysate at 30 µg

Lane 2:

Western blot - Human VCAM1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-vcam1-knockout-a549-cell-line-ab273758'>ab273758</a>)

Lane 3:

VCAM1 knockout A549 cell lysate at 30 µg

Lane 4:

VCAM1 knockout A549 TNF-a treated (10 ng/mL, 16h) cell lysate at 30 µg

Lane 5:

HUVEC cell lysate at 30 µg

Lane 6:

HUVEC TNF-a treated (16 ng/mL, 16h) cell lysate at 30 µg

Predicted band size: 81 kDa

Observed band size: 105 kDa

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