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Synthetic peptide corresponding to Human NF-kB p65. around the phosphorylation site of Serine 536.
Our Abpromise guarantee covers the use of ab86299 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||1/250 - 1/1000.|
|WB||1/2000 - 1/10000. Predicted molecular weight: 60 kDa.|
|IHC-P||1/500 - 1/2000.
Epitope retrieval with Tris-EDTA pH9.0 is recommended
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat lung tissue labeling NF-kB p65 (phospho S536) with ab86299 at 1/20 dilution. Tissue sections were fixed in 6%formalin and embedded into paraffin, 5 µm thin sections were cut with a microtome. Sections were stained with haematoxylin–eosin. Slides were deparaffinized in xilene, rehydrated in graded ethanol series, and washed in distilled water. Heat induced epitope retrieval was performed by boiling the tissue sections in citrate buffer in a microwave oven at 750 W followed by cooling at room temperature for 20 minutes. Slides were washed in tris buffered saline (TBS) solution (pH = 7,6) followed by blocking of endogenous peroxidase for 10 minutes at room temperature. Slides were washed in TBS. Nonspecific sites were blocked for 10 minutes at room temperature. Without washing, the primary antibody ab86299 was applied. Incubation with the primary antibody was performed for 1 hour at room temperature followed by washing in TBS. Anti-rabbit secondary antibody was applied for 30 minutes at room temperature followed by repeated washing in TBS.
IHC image of NF-kB p65 (phospho S536) staining in Human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. 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 ab86299, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer 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.
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