重组Anti-VCAM1抗体[EPR5047] - Low endotoxin，Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

This IHC data was generated using the same anti-VCAM1 antibody clone EPR5047 in a different buffer formulation (cat# ab134047).

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

This IHC data was generated using the same anti-VCAM1 antibody clone EPR5047 in a different buffer formulation (cat# ab134047).

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

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.

This data was developed using the same antibody clone in a different buffer formulation (ab271899).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

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).

• IP

Supplier Data

Immunoprecipitation - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

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='/products/primary-antibodies/vcam1-antibody-epr5047-ab134047'>ab134047</a>)

Predicted band size: 81 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

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).

• WB

Lab

Western blot - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

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='/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='/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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• WB

CiteAb

Western blot - Anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free (AB215380)

VCAM1 western blot using anti-VCAM1 antibody [EPR5047] - Low endotoxin, Azide free ab215380. Publication image and figure legend from Wu, F., Zhao, Y., et al., 2015, J Neuroinflammation, PubMed 25990934.

ab215380 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab215380 please see the product overview.

Astrocyte-derived CXCL1 and endothelial CXCR2 are essential for cerebral endothelial activation. A i.c.v. LPS injection (4 h) induced significant CXCR2 mRNA expression in WT mice. B Levels of CXCR2 protein after i.c.v. LPS injection from 4 to 24 h gradually increased compared with the control group (4 h after i.c.v. saline injection). C The expression of CXCR2 mRNA in primary brain microvascular endothelial cells, microglia, and astrocytes stimulated with either vehicle or TNF-α (100 ng/ml) was measured via real-time PCR. The results are represented as the means ± SEM of three independent experiments; *p < 0.05. D Primary endothelial cells, astrocytes, and microglia were seeded at 2 × 106 cells/well in six-well plates and were incubated overnight. The following day, the cells were stimulated with 100 ng/ml LPS or 100 ng/ml TNF-α for 12 h. Cell lysates were collected and analyzed for CXCR2 expression via Western blot. E Primary astrocytes and microglia from wild-type mice were seeded at 2 × 106 cells/well in six-well plates and were incubated overnight. The following day, the cells were stimulated with 100 ng/ml LPS or 100 ng/ml TNF-α for 12 h. Then, the conditioned supernatants and cell lysates were collected and analyzed for CXCL1 expression via ELISA. The results are represented as the means ± SEM of three independent experiments; **p < 0.01. F Astrocyte culture conditioned medium was added into primary cerebral endothelial cells from wild-type or CXCR2-/- mice, and the levels of VCAM-1 and ICAM-1 were measured via Western blot

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