Flow Cytometry analysis of TF-1 (human erythroleukemia) cells labeling CD34 with purified ab81289 at 1/50 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) (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) were used as the unlabeled control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling CD34 with purified ab81289 at 1/2500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as the secondary antibody at 1/500. Negative control using PBS instead of primary antibody. Counter stained with Hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of HUVEC (Human umbilical vein endothelial cell line) cells labelling CD34 with purified ab81289 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counter stain.

Control: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/500).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labelling CD34 with purified ab81289 at 1/2500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as the secondary antibody at 1/500. Negative control using PBS instead of primary antibody. Counter stained with Hematoxylin.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat lung tissue labeling CD34 with ab81289 at 1/50 (11.04 µg/mL) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Positive staining on rat lung. is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat kidney tissue labeling CD34 with ab81289 at 1/50 (11.04 µg/mL) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Positive staining on rat kidney. is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

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IHC image of CD34 staining in Mouse normal brain 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 ab81289, 1/250 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.

purified at 1/10000 dilution + TF-1 (Human bone marrow erythroleukemia cells) at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 41 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

CD34 was immunoprecipitated from TF-1 (Human bone marrow erythroleukemia cells) cells using purified ab81289 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab81289. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as secondary antibody at 1/1000 dilution. Blocking and dilution buffer and concentration: 5% NFDM/TBST

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Immunocytochemistry/Immunofluorescence analysis of human embryonic stem cell-derived endothelial cells labeling CD34 with unpurified ab81289 at 1/200. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% aTriton X-100. An Alexa Fluor^® 647-conjugated secondary antibody was used at a 1/400 dilution. DAPI (blue) was used as the nuclear counter stain.

Equilibrium disassociation constant (K_D)
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