Flow Cytometry - Anti-Cytokeratin 18 antibody [C-04] (ab668)

Overlay histogram showing HCT116 cells stained with ab668 (red line). The cells were fixed with 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 (ab668, 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 IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HCT116 cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Triton® used under the same conditions.

Immunocytochemistry/ Immunofluorescence - Cytokeratin 18 antibody [C-04] (ab668)

ab668 at 1/100 staining A431 Human epithelial carcinoma cells by ICC/IF. The cells were paraformaldehyde fixed, permeabilized with 0.25% Triton X-100 and then blocked with BSA prior to incubation with the antibody for 24 hours at 4°C. A FITC conjugated goat anti-mouse IgG antibody was used as the secondary. The right hand image shows staining with ab668 followed by secondary antibody. The left hand image shows DAPI staining with secondary only.

This image is courtesy of an anonymous Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Cytokeratin 18 antibody [C-04] (ab668)

ab668 (2µg/ml) staining cytokeratin 18 in human skin using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic staining of sweat coils.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Immunocytochemistry/ Immunofluorescence - Cytokeratin 18 antibody [C-04] (ab668)

ab668 staining Cytokeratin 18 in horse primary bronchial epithelial cells by Immunocytochemistry/ Immunofluorescence. The cells were fixed in acetone and then blocked using 3% BSA for 10 hours at 4ºC. Samples were then incubated with primary antibody at 1/100 for 1 hour at 22ºC. The secondary antibody used was a goat anti-mouse conjugated to FITC used at a 1/200 dilution.

Image courtesy of an anonymous Abreview.