ab20210 at 1/100 dilution staining Cytokeratin 19 breast cancer cell line by immunocytochemistry. Sections were methanol fixed prior to blocking in 10% serum for 5 minutes and then incubated with ab20210, for 1 hour at 25°C. Ultravision ONE HRP rabbit polyclonal to mouse Ig was used as the secondary antibody.

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ab20210 stained HepG2 cells. The cells were 100% methanol fixed for 5 minutes at room temperature and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab20210 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was ab150117 used at a 1/1000 dilution for 1hour at room temperature. The cells were counterstained with ab202277 (Rabbit monoclonal to alpha Tubulin (Alexa Fluor® 594)  (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

IHC image of Cytokeratin 19 staining in human normal liver formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ 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 ab20210, 5µg/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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : Anti-Cytokeratin 19 antibody [BA16] (ab20210) at 1 µg/ml

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 2 : Human liver tissue lysate - total protein (ab29889)
Lane 3 : Human tonsil normal tissue lysate - total protein (ab29615)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 40 kDa
Observed band size: 40,44 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute