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Synthetic peptide corresponding to Human Cyclin D1 (C terminal).
Our Abpromise guarantee covers the use of ab16663 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC||Use at an assay dependent concentration.|
|WB||1/25 - 1/200. Detects a band of approximately 36 kDa (predicted molecular weight: 33 kDa).|
|IHC-P||1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Deparaffinization: Deparaffinize slides using xylene or xylene alternative and graded alcohols.
Antigen Retrieval: Boil tissue section in 10mM citrate buffer, pH 6.0 for 10 min followed by cooling at room temperature for 20 min.
Primary Antibody Incubation: Incubate for 30 minutes at room temperature.
Slide Washing: Slides must be washed in between steps. Rinse slides with PBS/0.05% Tween.
ab16663 staining Cyclin D1 in MCF7 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab16663 at a working dilution of 1/250 and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594, shown in red) at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-rabbit AlexaFluor® 488 (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
IHC image of ab16663 staining Cyclin D1 in rat esophagus formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. 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 ab16663, 1:100 dilution, 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. No primary antibody was used in the secondary only control (shown on the inset).
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.
Image kindly supplied by Dr Karin Birkenkamp-Demtroeder through AbreviewGel run under denaturing conditions 4-12% gradient.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"