Anti-CXCR4 antibody [UMB2] - BSA and Azide free

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Characterization of UMB-2 (ab124824) by immunofluorescent staining of transfected cells. HEK-293 cells expressing CCR7 or CXCR4 were either not exposed or exposed to 100 ng/ml MIP-3 or 100 ng/ml SDF-1 for 30 min, subsequently fixed and immunofluorescently stained with 1 µg/ml anti-CCR7 {1188} or anti-CXCR4 {UMB-2} at a dilution of 1 : 100. Note that UMB-2 detected prominent immunofluorescence at the level of the plasma membrane only in CXCR4- but not in CCR7-expressing cells, and that SDF-1 exposure induced a rapid translocation of CXCR4 receptor immunostaining from the plasma membrane into the cytosol. Representative results from one of three independent experiments are shown. Scale bar, 20 µm.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124824).

Fischer, T. et al PLoS One. 2008;3(12):e4069. doi: 10.1371/journal.pone.0004069. Epub 2008 Dec 31 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124824).

Flow cytometry overlay histogram showing left Jurkat positive cells and right negative HepG2 stained with ab124824 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab124824) (1x 106 in 100μl at 1.0μg/ml (1/1900)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in Jurkat Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

This data was developed using ab124824, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of Paraffin-embedded sections Mouse spleen tissue labelling CXCR4 with ab124824 at 1/1000 dilution, followed by LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic and membranous staining on mouse spleen tissue is observed. Counter stained with Hematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

The section was incubated with ab124824 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

This data was developed using ab124824, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of Paraffin-embedded sections Mouse lung tissue labelling CXCR4 with ab124824 at 1/1000 dilution, followed by LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic and membranous staining on mouse lung tissue is observed. Counter stained with Hematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

The section was incubated with ab124824 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human B-cell non-hodgkin lymphoma tissue sections labeling CXCR4 with purified ab197203 at 1/1000 dilution (1.067 μg/ml). Heat mediated antigen retrieval was performed using Tris/EDTA Buffer, PH9 (ab93684). Hematoxylin was used to counter stain. Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used.

Cytoplasmic and membranous staining on tumor cells of human B-cell non-Hodgkin lymphoma.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Clone UMB2 (ab197203) has been successfully conjugated by Abcam. This image was generated using Anti-CXCR4 antibody [UMB2] (Alexa Fluor® 647). Please refer to ab208129 for protocol details.

ab208129 staining CXCR4 in Jurkat cells. The cells were fixed with 80% methanol (5min), 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 overnight at +4°C with ab208129 at 1/50 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in Jurkat cells fixed with 4% formaldehyde (10 min).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Unpurified ab124824, at 1/50 dilution, staining CXCR4 in paraffin-embedded Human tonsil tissue by immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124824).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Immunohistochemical detection of CXCR4 expression in human tissue specimens of normal appearance

CXCR4 was detected in the indicated PFA-fixed, paraffin-embedded human tissues using ab124824 at 5 μ/ml overnight at 4°C.

A, kidney. B, adrenal gland. C, cerebellum. D, bone marrow, brown staining : CXCR4, green staining : CD45. E, Spleen. F, testis. G, lung. H, colon.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124824).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Costa, M.J. et al PLoS One. 2018 Mar 19;13(3):e0194688. doi: 10.1371/journal.pone.0194688. eCollection 2018 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue sections labeling CXCR4 with purified ab197203 at 1/1000 dilution (1.067 μg/ml). Heat mediated antigen retrieval was performed using Tris/EDTA Buffer, PH9 (ab93684). Hematoxylin was used to counter stain. Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used.

Membranous with weak cytoplasmic staining on human tonsil.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124824). Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney) cells transfected with a mix of human CXCR1, CXCR2, CXCR3 and CXCR5 expression vector containing a myc-his tag (Left) / HEK-293T transfected with a human CXCR4 expression vector containing a myc-his tag (Right) labelling CXCR4 with ab124824 at 1/2000 dilution (0.1μg)/ Left and Right. A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/3000 dilution was used as the secondary antibody. No cross-reactivity with CXCR1/CXCR2/CXCR3/CXCR5.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Immunofluorescence staining of Jurkat cells with purified ab124824 at a working dilution of 1 in 250, counter-stained with DAPI. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor^® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The secondary antibody was ab150077 Alexa Fluor^® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in the bottom middle and right hand panels - for the first negative control, purified ab124824 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124824).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Intracellular Flow Cytometry analysis of Jurkat (human T cell leukemia T lymphocyte) cells labeling CXCR4 with purified ab124824 at 1/260 dilution (10 μg/ml) - Red. Cells were fixed with 4% paraformaldehyde . A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) - Black. Unlabeled control - Blue. Untreated cells - GreenThis data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab197203)

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

ab124824 stained Jurkat cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab124824 at 5ug/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124824).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Immunohistochemical staining of paraffin embedded human bladder cancer with purified ab124824 at a working dilution of 1/500. The secondary antibody used is ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is 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 (ab124824).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Unpurified ab124824, at 1/50 dilution, staining CXCR4 in paraffin-embedded Human cervical carcinoma tissue by immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124824).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Clone UMB2 (ab197203) has been successfully conjugated by Abcam. This image was generated using Anti-CXCR4 antibody [UMB2] (Alexa Fluor® 488). Please refer to ab208128 for protocol details.

ab208128 staining CXCR4 in Jurkat cells. The cells were fixed with 4% formaldehyde (10 min) and then incubated in 1%BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated overnight at +4°C with ab208128 at 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in Jurkat cells fixed with 80% methanol (5 min).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] - BSA and Azide free (AB197203)

Unpurified ab124824, at 1/50 dilution, staining CXCR4 in paraffin-embedded Human ovarian adenocarcinoma tissue by immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124824).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.