Anti-SHOX2 antibody [EPR30169-39]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MIA PaCa-2 (human pancreas epithelial cell) A431 (human epidermoid carcinoma epithelial cell) cells labelling SHOX2 with ab326071 at 1/200 (2.51 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing partial nuclear staining in MIA PaCa-2 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Low expression : A431. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab326071 at 1/200 dilution, followed by ab150120 at 1/1000 dilution.-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse DRG (Mouse primary dorsal root ganglion cell) cells labelling SHOX2 with ab326071 at 1/200 (2.51 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing mainly nuclear staining in mouse DRG (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

ab78078 Anti-beta III Tubulin mouse monoclonal antibody - Neuronal Marker was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab326071 at 1/200 dilution, followed by ab150120 at 1/1000 dilution.-ve control 2 : ab78078 at 1/500 dilution, followed by ab150081 at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunohistochemical analysis of paraffin-embedded Embryonic 14.5 rat tissue labeling SHOX2 with ab326071 at 1/500 (1.004 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on sinoatrial node of Embryonic 14.5 rat.

The primary antibody was incubated for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunohistochemical analysis of paraffin-embedded Embryonic 14.5 mouse tissue labeling SHOX2 with ab326071 at 1/500 (1.004 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on brain of Embryonic 14.5 mouse.

The primary antibody was incubated for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunohistochemical analysis of paraffin-embedded Embryonic 14.5 mouse tissue labeling SHOX2 with ab326071 at 1/500 (1.004 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on sinoatrial node of Embryonic 14.5 mouse (PMID : 18773496).

The primary antibody was incubated for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse splenocyte cells labelling SHOX2 with ab326071 at 1/200 (2.51 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Low expression : Confocal image showing weak staining in subset of mouse splenocytes (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-mouse CD3 rat monoclonal antibody (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• WB

Lab

Western blot - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : TT, A431.

The bands beneath the target band (33 kDa) are likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SHOX2 antibody [EPR30169-39] (ab326071) at 1/1000 dilution

Lane 1:

MIA PaCa-2 (human pancreas epithelial cell) transfected with scrambled siRNA control whole cell lysate at 50 µg

Lane 2:

MIA PaCa-2 transfected with siRNA specifically targeting SHOX2 whole cell lysate at 50 µg

Lane 3:

TT (human thyroid carcinoma epithelial cell) whole cell lysate at 50 µg

Lane 4:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 33-38 kDa,36 kDa

false

Exposure time: 114s

• WB

Lab

Western blot - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : TT.

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The bands beneath the target band (33 kDa) are likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SHOX2 antibody [EPR30169-39] (ab326071) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 50 µg

Lane 2:

TT (human thyroid carcinoma epithelial cell) whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 33-38 kDa,36 kDa

false

Exposure time: 70s

• WB

Lab

Western blot - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : testis, stomach, spleen.

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The identity of the higher MW band at approximately 50 kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SHOX2 antibody [EPR30169-39] (ab326071) at 1/1000 dilution

Lane 1:

Mouse E12 embryo tissue lysate at 50 µg

Lane 2:

Mouse E15 brain tissue lysate at 50 µg

Lane 3:

Mouse testis tissue lysate at 50 µg

Lane 4:

Mouse stomach tissue lysate at 50 µg

Lane 5:

Mouse spleen tissue lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 33-38 kDa,36 kDa

false

Exposure time: 70s

• WB

Lab

Western blot - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : pancreas, spleen.

The bands beneath the target band (33 kDa) are likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SHOX2 antibody [EPR30169-39] (ab326071) at 1/1000 dilution

Lane 1:

Rat E15 brain tissue lysate at 50 µg

Lane 2:

Rat embryo tissue lysate at 50 µg

Lane 3:

Rat spleen tissue lysate at 50 µg

Lane 4:

Rat pancreas tissue lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 33-38 kDa,36 kDa

false

Exposure time: 70s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling SHOX2 with ab326071 at 1/500 (1.004 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining on mouse spleen.

The primary antibody was incubated for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling SHOX2 with ab326071 at 1/500 (1.004 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining on mouse kidney.

The primary antibody was incubated for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T transfected with a mouse Shox2 expression vector containing a His tag (B) HEK-293T transfected with an empty expression vector containing a His tag tissue labeling SHOX2 with ab326071 at 1/2000 (0.251 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) HEK-293T transfected with a mouse Shox2 expression vector containing a His tag cell pellets, no staining on (B) HEK-293T transfected with an empty expression vector containing a His tag cell pellet.

The primary antibody was incubated for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling SHOX2 with ab326071 at 1/500 (1.004 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining on rat spleen.

The primary antibody was incubated for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling SHOX2 with ab326071 at 1/500 (1.004 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining on rat kidney.

The primary antibody was incubated for 30 mins at room temperature.

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

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• Dot

Lab

Dot Blot - Anti-SHOX2 antibody [EPR30169-39] (AB326071)

Dot blot analysis of SHOX2 using ab326071 at 1 : 1000 (0.502 µg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane 1 : His-tagged human SHOX fragment

Lane 2 : His-tagged human SHOX2 fragment

Exposure time : 180 seconds.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody does not cross-react with human SHOX.

All lanes:

Dot Blot - Anti-SHOX2 antibody [EPR30169-39] (ab326071) at 1/1000 dilution

Lane 1:

His-tagged human SHOX fragment

Lane 2:

His-tagged human SHOX2 fragment

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 180s