Immunofluorescent analysis of 4% Paraformaldehyde, 0.1% Triton X-100 permeabilized PC12 (Rat adrenal gland pheochromocytoma) cells, labeling Cytokeratin 18 with ab181597 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasm staining on PC-12 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab181597 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Immunohistochemical analysis of paraffin-embedded Human colonic adenocarcinoma tissue labeling Cytokeratin 18 with ab181597 at 1/800 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. IHC showed membrane and cytoplasm staining on tumor cells of Human colon cancer. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 2% paraformaldehyde-fixed MCF7 (Human breast adenocarcinoma cell line) cells labeling Cytokeratin 18 with ab181597 at 1/120 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and a unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Cytokeratin 18 with ab181597 at 1/800 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. IHC showed membrane and cytoplasm staining on hepatocytes of Human liver. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Cytokeratin 18 with ab181597 at 1/800 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. IHC showed membrane and cytoplasm staining on hepatocytes of mouse liver. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling Cytokeratin 18 with ab181597 at 1/800 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. IHC showed membrane and cytoplasm staining on tubules of rat kidney. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized GR-M (Human Caucasian melanoma) cells, labeling Cytokeratin 18 with ab181597 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasm staining on GR-M cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab181597 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Immunofluorescent analysis of 100% Methanol, 0.1% Triton X-100 permeabilized 4T-1 (Mouse mammary gland carcinoma cell line) cells, labeling Cytokeratin 18 with ab181597 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasm staining on 4T-1 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
1. ab181597 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

All lanes : Anti-Cytokeratin 18 antibody [EPR17347] (ab181597) at 1/10000 dilution

Lane 1 : Mouse colon lysate
Lane 2 : Rat lung lysate
Lane 3 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa
Observed band size: 45,48 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

5% NFDM/TBST: Blocking and diluting buffer.

The observed MW is consistent with what has been described in the literature (PMID:9298992).

All lanes : Anti-Cytokeratin 18 antibody [EPR17347] (ab181597) at 1/5000 dilution

Lane 1 : Mouse kidney lysate
Lane 2 : Mouse spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa
Observed band size: 45,48 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

5% NFDM/TBST: Blocking and diluting buffer.

The observed MW is consistent with what has been described in the literature (PMID:9298992).

Anti-Cytokeratin 18 antibody [EPR17347] (ab181597) at 1/10000 dilution + Human fetal skin lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 47 kDa
Observed band size: 45,48 kDa why is the actual band size different from the predicted?


Exposure time: 2 minutes

5% NFDM/TBST: Blocking and diluting buffer.

The observed MW is consistent with what has been described in the literature (PMID:9298992).

Anti-Cytokeratin 18 antibody [EPR17347] (ab181597) at 1/2000 dilution + F9 (Mouse embyro testicular cancer cell line) whole cell lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 47 kDa
Observed band size: 45,48 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

The observed MW is consistent with what has been described in the literature (PMID:9298992).

All lanes : Anti-Cytokeratin 18 antibody [EPR17347] (ab181597) at 1/5000 dilution

Lane 1 : Untreated HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa whole cell lysate treated with 1uM staurosporine for 4 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa
Observed band size: 22,45,48 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

5% NFDM/TBST: Blocking and diluting buffer.

The 22 kDa cytokeratin 18 fragment was generated (lane 2) in the staurosporine-treated lysate that contained the active caspase 3.

All lanes : Anti-Cytokeratin 18 antibody [EPR17347] (ab181597) at 1/5000 dilution

Lane 1 : Untreated HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa whole cell lysate treated with 50uM Z-VAD-FMK for 4 hours

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 47 kDa
Observed band size: 45,48 kDa why is the actual band size different from the predicted?


Exposure time: 2 seconds

5% NFDM/TBST: Blocking and diluting buffer.

The generation of the 22kDa fragment was inhibited when the caspase 3 activity was blocked by the caspase inhibitor, Z-VAD-FMK.