Anti-GPA33 antibody [EPR30348-518]

• mIHC

Lab

Multiplex immunohistochemistry - Anti-GPA33 antibody [EPR30348-518] (AB324562)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse colon tissue staining GPA33 with ab324562 at a 1/100 dilution, ab324560 anti-ATAD2 used at 1/500 dilution and ab272692 anti-MUC2 used at a 1/2000 dilution.

Panel A : anti-GPA33 (green; Opal™690), anti-ATAD2 (magenta; Opal™520) and anti-MUC2 (gray; Opal™570) on mouse colon.

Panel B : anti-GPA33 staining membrane of epithelium in mouse colon.

Panel C : anti-ATAD2 staining nucleus of proliferating cells in mouse colon.

Panel D : anti-MUC2 staining goblet cells in mouse colon.

Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab324562, ab324560 and ab272692 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GPA33 antibody [EPR30348-518] (AB324562)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling GPA33 with ab324562 at 1/100 (5.03 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining on mouse spleen (PMID : 32678100).
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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GPA33 antibody [EPR30348-518] (AB324562)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling GPA33 with ab324562 at 1/100 (5.03 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining on mouse liver (PMID : 32678100).
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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-GPA33 antibody [EPR30348-518] (AB324562)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Tween-20 permeabilized 4T1(mouse mammary gland carcinoma epithelial cell) cells labelling GPA33 with ab324562 at 1/50 (10.06 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Confocal image showing membranous staining in 4T1 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : B16-F10.
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/ab7291 1000 1ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab324562 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-GPA33 antibody [EPR30348-518] (AB324562)

Flow cytometric analysis of B16-F10 (mouse skin melanoma cell, Left) / 4T1 (mouse mammary gland carcinoma epithelial cell, Right) cells labelling GPA33 with ab324562 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Low expression : B16-F10.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GPA33 antibody [EPR30348-518] (AB324562)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling GPA33 with ab324562 at 1/100 (5.03 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon.
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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GPA33 antibody [EPR30348-518] (AB324562)

Immunohistochemical analysis of paraffin-embedded Mouse colon cancer tissue labeling GPA33 with ab324562 at 1/100 (5.03 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon cancer.
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

• WB

Supplier Data

Western blot - Anti-GPA33 antibody [EPR30348-518] (AB324562)

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

Low expression : B16-F10.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-GPA33 antibody [EPR30348-518] (ab324562) at 1/1000 dilution

Lane 1:

4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

B16-F10 (mouse skin melanoma cell) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 43 kDa,36 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-GPA33 antibody [EPR30348-518] (AB324562)

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

Negative control : skeletal muscle, kidney, liver, heart, lung, spleen (PMID : 27721471).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-GPA33 antibody [EPR30348-518] (ab324562) at 1/1000 dilution

Lane 1:

Mouse small intestine tissue lysate at 20 µg

Lane 2:

Mouse colon tissue lysate at 20 µg

Lane 3:

Mouse kidney tissue lysate at 20 µg

Lane 4:

Mouse liver tissue lysate at 20 µg

Lane 5:

Mouse heart tissue lysate at 20 µg

Lane 6:

Mouse lung tissue lysate at 20 µg

Lane 7:

Mouse spleen tissue lysate at 20 µg

Lane 8:

Mouse skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 43 kDa,36 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-GPA33 antibody [EPR30348-518] (AB324562)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-6X His tag® antibody [AD1.1.10] - (ab15149) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-GPA33 antibody [EPR30348-518] (ab324562) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 2:

293T cells transfected with a mouse GPA33 expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Secondary

All lanes:

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

Observed band size: 43 kDa,36 kDa

false

Exposure time: 3s