Recombinant 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).

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).

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).

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.

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).

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).

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).

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.

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.

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.

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.

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.