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Our Abpromise guarantee covers the use of ab18259 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 50 kDa (predicted molecular weight: 50 kDa).|
|ChIP||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 0.5 - 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|ICC/IF||Use at an assay dependent concentration.|
IHC image of FoxG1 staining in mouse brain E14 formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab18259, 0.5 µg/ml, 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.
Staining revealed in telencephalon (but not diencephalon) as expected.
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.
ab18259 staining FOXG1 in Rat embryonic cortex cells overexpressing FOXG1 by ICC/IF (Immunocytochemistry/ immunofluorescence). Cells were fixed with formaldehyde and blocked with 1o% serum for 1 hour at 25°C. Samples were incubated with primary antibody (1/500) for 24 hours at 4°C. A Cy3®-conjugated anti-rabbit IgG polyclonal was used as the secondary antibody (1/500).
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab18259 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
ab18259 detects a band of ~50kDa in brain lysates. This band is blocked by addition of the immunizing peptide ab19644 which suggests that is specific for FOXG1.
This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab18259 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody, and visualised using ECL development solution.
ab18259 staining Foxg1 (1/50) in the telencephalon (but not the diencephalon) as expected. DAB-immunohistochemistry was performed on embryonic (E13.5) mouse brain (coronal paraffin-embedded sections) after microwave treatment with 10mM sodium citrate.
ab18259 staining FOXG1 in human A549 cells by immunocytochemistry/immunofluorescence.Cells were fixed in paraformaldehyde, permeabilized using 0.25% Triton, blocked with 1% BSA for 1 hour at room temperature and then incubated with ab18259 at a 1/400 dilution for 1 hour. The secondary used was an Alexa-Fluor 488 conjugated goat polyclonal used at a 1/250 dilution.