Alexa Fluor® 647 Anti-LEF1 antibody [EPR2029Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-LEF1 antibody [EPR2029Y] (AB246715)

Overlay histogram showing Jurkat cells stained with ab246715 (red line). The cells were fixed with 80 % methanol (5 min) and then permeabilized with 0.1 % PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10 % normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab246715) (1x 10⁶ in 100μl at 0.01 μg/ml (1/50000)) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Alexa Fluor^® 647 (ab199093) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 40 mW Red laser (640nm) and 670/14 bandpass filter.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 647 Anti-LEF1 antibody [EPR2029Y] (AB246715)

ab246715 staining LEF1 in HT-29 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 ab246715 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Alexa Fluor® 647 Anti-LEF1 antibody [EPR2029Y] (AB246715)

IHC image of LEF1 staining in a section of formalin-fixed paraffin-embedded normal human tonsil*.

The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer (pH9, epitope retrieval solution 2) for 20mins, performed on a Leica BOND^™. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab246715 at 1/100 dilution (shown in red) and counterstained using ab202272, Rabbit monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in green). Nuclear DNA was labeled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.