IHC image of Cytokeratin 14 staining in a section of formalin-fixed paraffin-embedded normal human skin* performed on a Leica BOND^TM 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 20mins. The section was then incubated with ab7800, 1ug/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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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

ab7800 staining KRT14 in wild-type A431 cells (top panel) and KRT14 knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab7800 at 1μg/ml concentration and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor^® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor^® 594) (ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

All lanes :

Lane 1 : A431 whole cell lysate
Lane 2 : Human skin whole tissue lysate
Lane 3 : SH-SY5Y whole cell lysate (negative control)

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 55 kDa why is the actual band size different from the predicted?
Additional bands at: 70 kDa (possible cross reactivity)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before ab7800 and ab181602 (Rabbit anti GAPDH), were incubated overnight at 4°C at a 1ug/ml concentration and 1/20000 dilution respectively. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab7800 staining Cytokeratin 14 in A431 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab7800 at 0.1ugml then detected with an Alexa Fluor^® 488 goat anti-mouse secondary antibody (ab150117) at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab202272, Rabbit monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red).

ab7800 staining Cytokeratin 14 in Human normal skin tissue sections by IHC-P (Formaldehyde-fixed, Paraffin-embedded sections). Tissue samples were fixed with formaldehyde and blocked with 10% Serum for 30 minutes at 21°C; antigen retrieval was by heat mediation in citrate buffer (pH 6). The sample was incubated with primary antibody (1/100 in PBS + 0.5% Tween-20 + 0.5% BSA)) at 21°C for 30 minutes. An undiluted HRP-conjugated goat polyclonal to mouse IgG was used as secondary antibody.

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Anti-Cytokeratin 14 antibody [LL002] (ab7800) at 2 µg/ml + Human HaCaT whole cell lysate at 30 µg

Secondary
Goat Anti-mouse IgG Polyclonal at 1/20000 dilution

Developed using the ECL technique.

Observed band size: 55 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute


Blocking Step: 5% Milk for 12 hours at 4°C
Gel Running Conditions: 15%,6V,50min; Reduced; Denaturing

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