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Our Abpromise guarantee covers the use of ab14683 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|WB||1/2500. Detects a band of approximately 50 kDa (predicted molecular weight: 55 kDa). We suggest that samples should not be heated or frozen.|
|IP||Use at an assay dependent concentration.|
Human normal placenta. Staining is observed at the cell surface.
Left panel: Primary antibody at 2 ug/ml.
Right panel: Isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH 6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutess. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako envision flex amplification kit for rabbit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with haematoxylin and coverslipped under DePeX.
Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
ICC/IF image of ab14683 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. The cells were 4% formaldehyde fixed (10 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14683, 5 µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.
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