Anti-IL13RA2 antibody [RM2087]

• Flow Cyt

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Flow Cytometry - Anti-IL13RA2 antibody [RM2087] (AB324598)

Flow cytometric analysis of IL13RA2 KO A375 (IL13RA2 knockout human malignant melanoma epithelial cell) (Magenta) / A375 (Green) cells labelling IL13RA2 with ab324598 at 1/50 dilution (1ug) / Green and magenta compared with a Rabbit monoclonal IgG (ab172730) (Black) and Grey 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL13RA2 antibody [RM2087] (AB324598)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling IL13RA2 with ab324598 at 1/100 (5.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining on human testis. 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) was used.

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL13RA2 antibody [RM2087] (AB324598)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling IL13RA2 with ab324598 at 1/100 (5.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining on immune cells of human colon carcinoma. 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) was used.

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IL13RA2 antibody [RM2087] (AB324598)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized IL13RA2 KO A375(IL13RA2 knockout human malignant melanoma epithelial cell), ab273381 cells labelling IL13RA2 with ab324598 at 1/1000 (0.505 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green).

Confocal image showing membranous and cytoplasmic staining in wiltype A375 cells (shown in green), showing no staining in IL13RA2 knockout A375 cells. 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).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1ug/ml) 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 (2ug/ml) dilution.

• Flow Cyt

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Flow Cytometry - Anti-IL13RA2 antibody [RM2087] (AB324598)

Flow cytometric analysis of A-172 (human brain glioblastoma cell) (Right) / Daudi (human Burkitt's lymphoma lymphoblast) (Left) cells labelling IL13RA2 with ab324598 at 1/50 dilution (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.
Negative control : Daudi.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL13RA2 antibody [RM2087] (AB324598)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type A375 (human malignant melanoma epithelial cell) cell pellet (B) IL13RA2 knockout A375 cell pellet tissue labeling IL13RA2 with ab324598 at 1/100 (5.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining on (A) Wild-type A375 cell pellet, no staining on (B) IL13RA2 knockout A375 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) was used.

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL13RA2 antibody [RM2087] (AB324598)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling IL13RA2 with ab324598 at 1/100 (5.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Negative control : no staining on human skeletal muscle. 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) was used.

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

• WB

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Western blot - Anti-IL13RA2 antibody [RM2087] (AB324598)

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

Performed under reducing conditions.

In Western blot, ab324598 was shown to bind specifically to IL-13 receptor alpha 2. Band of interest was observed at 50-60 kDa in wild-type A375 cell lysate (lane 1) with no signal observed at this size in IL13RA2 knockout A375 cell lysate (lane 2) (lane 2, knockout cell line ab273381 / knockout cell lysate ab275532).

The molecular weight observed is consistent with what has been described in the literature (PMID : 30718742).

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

Exposure time : Lanes 1-2 : 26 seconds, lane 3 : 48 seconds, lane 4 : 180 seconds

All lanes:

Western blot - Anti-IL13RA2 antibody [RM2087] (ab324598) at 1/1000 dilution

Lane 1:

A375 (human malignant melanoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Western blot - Human IL13RA2 knockout A375 cell lysate (<a href='/en-us/products/cell-lysates/human-il13ra2-knockout-a375-cell-lysate-ab275532'>ab275532</a>) at 20 µg

Lane 3:

A-172 (human brain glioblastoma cell) whole cell lysate at 20 µg

Lane 4:

Human testis tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 50-60 kDa,36 kDa

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