重组Anti-LEF1抗体[EPR2029Y] (ab137872)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling LEF1 with ab137872 at a concentration of 0.5 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab137872 anti LEF1 antibody [EPR2029Y] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Flow cytometry overlay histogram showing left Jurkat positive cells and right negative HeLa stained with ab137872 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab137872) (1x 10⁶ in 100μl at 0.2μg/ml (1/11500)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in Jurkat Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

False colour image of Western blot: Anti-LEF1 antibody [EPR2029Y] staining at 1/1000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab137872 was shown to bind specifically to LEF1. A band was observed at 40/53 kDa in wild-type Jurkat cell lysates with no signal observed at this size in Lef1 knockout cell line ab274898 (knockout cell lysate ab274956). To generate this image, wild-type and Lef1 knockout Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with Optiblot (ECL reagent ab133456) and imaged with 4 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human tonsil with purified ab137872 at a working dilution of 1/500. The secondary antibody used is ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunofluorescence staining of Jurkat (Human T cell leukemia cell line from peripheral blood) cells with purified ab137872 at a working dilution of 1 in 500, counter-stained with DAPI. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor^® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The secondary antibody was ab150077 Alexa Fluor^® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100.

The negative controls are shown in the bottom middle and right hand panels - for the first negative control, purified ab137872 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

Intracellular flow cytometric analysis of Jurkat cell line (human T cell leukemia T lymphocyte) fixed with 4% paraformaldehyde and permeabilized with 90% methanol labeling LEF1 with ab137872 at 1/600 dilution (red). This is compared with a Rabbit monoclonal IgG (ab172730) - Isotype control (black) and a unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti-rabbit IgG (Alexa Fluor^®488) was used as the secondary antibody. 

Lane 1 (input): Jurkat (human T cell leukemia T lymphocyte) whole cell lysate, 10 μg
Lane 2 (+): Jurkat whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of  ab137872 in Jurkat  whole cell lysate

ab137872 immunoprecipitating LEF1 in Jurkat whole cell lysate. For western blotting, primary antibody used was ab137872 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST

Exposure time: 3 minutes

Immunohistochemical staining of paraffin embedded rat spleen with purified ab137872 at a working dilution of 1/500. The secondary antibody used is ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin-embedded human thymus with purified ab137872 at a working dilution of 1/500. The secondary antibody used is ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Tissue Microarrays stained for Anti-LEF1 antibody [EPR2029Y] using ab137872 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. The sections were incubated with ab137872 for 30 mins at room temperature used at 1:2000 dilution (1.05 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB secondary antibody (ab209101). Counterstain was Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

ab137872 staining LEF1 in paraffin embedded human thymona tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.  Samples were incubated with primary antibody at 1/2000 dilution for 30 mins at room temperature. A ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

ab137872 staining LEF1 in paraffin embedded human melanoma tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.  Samples were incubated with primary antibody at 1/2000 dilution for 30 mins at room temperature. A ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

ab137872 staining LEF1 in paraffin embedded mouse spleen tissue by Immunohistochemistry. Antigen retrieval was performed by heat mediation using ab93684 (Tris/EDTA buffer, ph 9).  Samples were incubated with primary antibody at 1/2000 dilution. A ready to use Goat Anti-rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain. Nuclear staining on T cells in periarterial lymphatic sheath of mouse spleen is observed (PMID: 21685909).

Blocking/Dilution buffer: 5% NFDM/TBST

Blocking/Dilution buffer: 5% NFDM/TBST

Blocking and diluting buffer: 5% NFDM/TBST

Lane 1 (input): Rat thymus lysate, 10μg
Lane 2 (+): Rat thymus lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of  ab137872 in rat thymus lysate

ab137872 immunoprecipitating LEF1 in rat thymus lysate. For western blotting, primary antibody used was ab137872 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST

Exposure time: 3 minutes