Biotin Anti-TTF1/Nkx2-1 antibody [EP1584Y]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Biotin Anti-TTF1/Nkx2-1 antibody [EP1584Y] (AB314937)

Negative staining of TTF1/Nkx2-1 in a section of formalin-fixed paraffin-embedded mouse normal skin.

Performed on a Leica BOND^TMsystem. The section was pre-treated using heat mediated antigen retrieval with EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins, before blocking of endogenous biotin using ab64212. The section was then incubated with ab314937 at 5 ug/ml, for 15 mins at room temperature and detected using an HRP conjugated ABC 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Biotin Anti-TTF1/Nkx2-1 antibody [EP1584Y] (AB314937)

IHC image of TTF1/Nkx2-1 staining in a section of formalin-fixed paraffin-embedded mouse normal lung.

Performed on a Leica BOND^TMsystem. The section was pre-treated using heat mediated antigen retrieval with EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins, before blocking of endogenous biotin using ab64212. The section was then incubated with ab314937 at 5 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated ABC 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.

• WB

Lab

Western blot - Biotin Anti-TTF1/Nkx2-1 antibody [EP1584Y] (AB314937)

Western blot : Anti-TTF1/Nkx2-1 antibody [EP1584Y] (ab314937) staining at 1/2000 dilution, shown in black; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta.

In Western blot, ab314937 was shown to bind specifically to NKX2-1/TTF-1. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% 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 a high-sensitivity ECL substrate kit.

Secondary antibodies used were HRP conjugated Streptavidin at 0.1 µg/ml and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Biotin Anti-TTF1/Nkx2-1 antibody [EP1584Y] (ab314937) at 1/2000 dilution

Lane 1:

Hela cell lysate at 20 µg

Lane 2:

HEK-293 cell lysate at 20 µg

Secondary

Lane 1:

HRP conjugated Streptavidin at 0.1 µg/mL

Lane 2:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 39 kDa,75 kDa

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Exposure time: 20min