The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration.
Use 1µg for 106 cells.
ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
Expressed in a discontinuous manner in the basal cell layer of adult skin epidermis, but continuously in the basal layer of fetal skin epidermis and nail. Also expressed in the outer root sheath above the hair bulb in hair follicle (at protein level). Expressed homogeneously in all cell layers of the esophagus and exocervix, but detected in the basal cell layer only of oral mucosa, skin and in the basal plus the next two layers of the suprabasal epithelium of the palate.
IHC image of Cytokeratin 15 staining in a formalin fixed, paraffin embedded normal human breast 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 ab80522, 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
ab80522 stained in A431cells. The cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1 hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab80522 at 5 µg/ml and ab6046 (Rabbit polyclonal to beta tubulin) at 1ug/ml overnight at +4°C. The secondary antibodies were ab150117 (colored green) used at 1 ug/ml and ab150087 (pseudo-colored red) used at 2 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 15 antibody [LHK15] - BSA and Azide free (ab80522)This image is courtesy of an anonymous Abreview
ab80522 staining Cytokeratin 15 in mouse colon tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde; antigen retrieval was by heat mediation. Samples were incubated with primary antibody (undiluted) for 10 hours.
Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 15 antibody [LHK15] (ab80522)This image is courtesy of an Abreview submitted by Maciej Gagat
ab8969 staining keratin 15 in rat hair folicles by immunocytochemistry/ immunofluorescence. Cells were paraformaldehyde fixed and permeabilized in 0.25% Triton X-100 prior to blocking in 1% BSA for 10 minutes at 21°C. The primary antibody was diluted 1/100 and incubated with the sample for 1 hour at 21°C. A TRITC conjugated mouse anti-mouse antibody, diluted 1/85, was used as the secondary.
Overlay histogram showing A431 cells stained with ab80522 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab80522, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.