Anti-Cytokeratin 8 antibody [M20] (ab9023)

Western blot - Cytokeratin 8 antibody [M20] (ab9023)

The image shows different concentration of total protein from normal and tumour colon tissues.

This picture was kindly supplied as part of the review submitted by Semona Rupchand.

Immunocytochemistry/ Immunofluorescence - Cytokeratin 8 antibody [M20] (ab9023)

ab9023 staining MCF-7 breast cancer cells, grown in 8 well chamber slides, by ICC/IF.  Cells were fixed with Acetone for 20 minutes at -20°C and permabilized with 0.1% Triton X-100 for 5 minutes at 4°C prior to blocking in 1% fish gelatin for 1 hour at RT.  The primary antibody was diluted 1/100 and incubated with the sample for 1 hour.  An Alexa Fluor® 488 conjugated donkey anti-mouse antibody diluted 1/1000 was used as the secondary.  Slides were mounted using mounting medium containing DAPI.

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - Cytokeratin 8 antibody [M20] (ab9023)

Ab9023 staining cells from a human pancreatic cancer cell line by ICC/IF.  The cells were PFA fixed and permeabilized in 0.1% Triton X-100 prior to blocking in 1% BSA for 30 minutes at 22°C.  Ab9023 was diluted 1/100 and incubated with the sample for 1 hour at 22°C.  An Alexa Fluor® 488 conjugated donkey anti-mouse antibody, diluted 1/1000, was used as the secondary.

This image is courtesy of an Abreview submitted by Dr Hannah Whiteman

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Cytokeratin 8 antibody [M20] (ab9023)

Ab9023 staining human normal liver parenchima tissue. Staining is localized to cell membrane.
Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes 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 we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Western blot - Cytokeratin 8 antibody [M20] (ab9023)

All lanes : Anti-Cytokeratin 8 antibody [M20] (ab9023) at 1/2000 dilution

Lane 1 : Lysate prepared from human prostate cancer LNCaP cells
Lane 2 : Lysate prepared from human prostate cancer PC3 cells

Lysates/proteins at 50 µg per lane.

Secondary
HRP-conjugated goat polyclonal to mouse IgG at 1/10000 dilution
developed using the ECL technique

Performed under reducing conditions.

Observed band size : 50 kDa (why is the actual band size different from the predicted?)
Additional bands at : 26 kDa,65 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 4 minutes

This image is a courtesy of Anonymous Abreview

Flow Cytometry - Cytokeratin 8 antibody [M20] (ab9023)

Overlay histogram showing MCF-7 cells stained with ab9023 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.5% 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 followed by the antibody (ab9023, 2µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H&L) (ab96879) at 1/250 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 MCF-7 cells fixed with methanol (5 min)/permeabilized in 0.5% PBS-Tween used under the same conditions.

Immunocytochemistry/ Immunofluorescence - Cytokeratin 8 antibody [M20] (ab9023)

ab9023 staining Cytokeratin 8 in human epithelial ovarian cancer (EOC) cell line OV-MZ-6 by Immunocytochemistry/ Immunofluorescence.

Samples were fixed with 4% PFA in PBS pH 7.4 and then permeabilised using 0.2% saponin for 30 minutes. A blocking step was performed using 1% BSA/PBS for 1 hour. Samples were then incubated with ab9023 at a 1/100 dilution in 1% BSA/PBS for 1 hour. The secondary antibody was a goat anti-mouse Alexa 488 (green) diluted 1/1000, 1% BSA/PBS for 1 hour. Samples were then incubated with phalloidin (red for actin staining) in 1% BSA/PBS for 45 minutes and counterstained with DAPI (blue for nuclei staining) in PBS for 45 minutes.

Image from Loessner D et al, Biomaterials. 2010 Nov;31(32):8494-506. Epub 2010 Aug 14. doi:10.1016/j.biomaterials.2010.07.064