Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - pan Keratin antibody [80] (ab8068)

ab8068 staining human foreskin tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin embedded). Tissue underwent fixation in paraformaldehyde, heat mediated antigen retrieval in 10mM Citrate buffer pH 6.0 in boiling water bath for 10 minutes, followed by cooling at room temperature for 30 minutes. The blocking was done using 1%BSA/5% normal donkey serum in PBS pH 7.4 for 2 hours at room temperature. The primary antibody, diluted 1/10 (PBS pH 7.4, 1%BSA, 0.1% sodium azide) and incubated with sample for 16 hours at 4°C. A HRP-conjugated donkey polyclonal to mouse Ig, diluted 1/1000 was used as the secondary.

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence-pan Keratin antibody [80](ab8068)

ICC/IF image of ab8068 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8068, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Western blot - pan Keratin antibody [80] (ab8068)

Anti-pan Keratin antibody [80] (ab8068) at 1/250 dilution + A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 65 kDa
Observed band size : 65 kDa
Additional bands at : 49 kDa,58 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 30 seconds

Flow Cytometry-pan Keratin antibody [80](ab8068)

Overlay histogram showing A431 cells stained with ab8068 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 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 followed by the antibody (ab8068, 1/20 dilution) 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 IgG1 [ICIGG1](ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Immunocytochemistry/ Immunofluorescence - Anti-pan Keratin antibody [80] (ab8068)

ab8068 staining pan Keratin in rat glioblastoma cell line C6 by Immunocytochemistry/ Immunofluorescence.

Cells were fixed in paraformaldehyde, permeabilized using 0,1% Triton X 100 in PBS, blocked with 0.5% BSA for 30 minutes at room temperature and then incubated with ab8068 at a 1/50 dilution for 16 hours at 4°C. The secondary used was a Cy3 conjugated goat anti-mouse polyclonal used at a 1/400 dilution. Nuclei are counterstained with DAPI.

Image courtesy of an anonymous Abreview.