Anti-ITPR2 antibody [EPR28319-63]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling ITPR2 with ab320725 at 1/2000 (0.266 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat cerebrum. The section was incubated with ab320725 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-Fr

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Immunohistochemistry (Frozen sections) - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (fresh frozen) tissue labeling ITPR2 with ab320725 at 1/50 (10.62 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Panel A : merged staining of anti-ITPR2 (ab320725, magenta), anti-Olig2 (ab225099, green) and anti-GFAP (ab201732, grey) on mouse cerebrum.
Panel B : anti-ITPR2 stained on mouse cerebrum.
Panel C : anti-Olig2 stained in oligodendrocyte of mouse cerebrum.
Panel D : anti-GFAP stained in astrocytes of mouse cerebrum.
The section was incubated in two rounds of staining : in the order of ab320725 and ab225099, ab201732 for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cardiac muscle (fresh frozen) tissue labeling ITPR2 with ab320725 at 1/50 (10.62 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Low expression : confocal image showing no staining on rat cardiac muscle. The nuclear counterstain was DAPI (Blue). The section was incubated with ab320725 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling ITPR2 with ab320725 at 1/2000 (0.266 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on mouse cardiac muscle. The section was incubated with ab320725 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-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cardiac muscle (fresh frozen) tissue labeling ITPR2 with ab320725 at 1/50 (10.62 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Low expression : confocal image showing no staining on mouse cardiac muscle. The nuclear counterstain was DAPI (Blue). The section was incubated with ab320725 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum (fresh frozen) tissue labeling ITPR2 with ab320725 at 1/50 (10.62 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Panel A : merged staining of anti-ITPR2 (ab320725, magenta), anti-Olig2 (ab225099, green) and anti-GFAP (ab201732, grey) on rat cerebrum.
Panel B : anti-ITPR2 stained on rat cerebrum.
Panel C : anti-Olig2 stained in oligodentrocytes of rat cerebrum.
Panel D : anti-GFAP stained in astrocytes of rat cerebrum.
The section was incubated in two rounds of staining : in the order of ab320725 and ab225099, ab201732 for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling ITPR2 with ab320725 at 1/2000 (0.266 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum (PMID : 26433454). The section was incubated with ab320725 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

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Western blot - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

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

Low expression : human spleen, mouse heart, rat heart (PMID : 7744807).

Samples are non-boiled as boiling may cause protein aggregation.

Lanes 1&2 are applied with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 and Lanes 3-8 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-ITPR2 antibody [EPR28319-63] (ab320725) at 1/1000 dilution

Lane 1:

Human brainstem whole tissue lysate, in the loading buffer containing double DTT at 40 µg

Lane 2:

Human spleen whole tissue lysate, in the loading buffer containing double DTT at 40 µg

Lane 3:

Mouse brain whole tissue lysate, in the loading buffer containing double DTT at 40 µg

Lane 4:

Mouse liver whole tissue lysate, in the loading buffer containing double DTT at 40 µg

Lane 5:

Mouse heart whole tissue lysate, in the loading buffer containing double DTT at 40 µg

Lane 6:

Rat brain whole tissue lysate, in the loading buffer containing double DTT at 40 µg

Lane 7:

Rat liver whole tissue lysate, in the loading buffer containing double DTT at 40 µg

Lane 8:

Rat heart whole tissue lysate, in the loading buffer containing double DTT at 40 µg

Secondary

Lanes 1 - 2:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG (Merck DC03L) at 1/2000 dilution

Lanes 3 - 8:

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 1/100000 dilution

Observed band size: 260 kDa,124 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

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

Samples are non-boiled as boiling may cause protein aggregation.

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 (Lane 8&9).

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

Exposure time : Lane 1-7 : 114 seconds; Lane 8 : 26 seconds; Lane 9 : 7 seconds

All lanes:

Western blot - Anti-ITPR2 antibody [EPR28319-63] (ab320725) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control, whole cell lysate, in the loading buffer containing double DTT at 20 µg

Lane 2:

HeLa transfected with siRNA specifically targeting ITPR2, whole cell lysate, in the loading buffer containing double DTT at 40 µg

Lane 3:

HepG2 (human hepatocelullar carcinoma epithelial cell), whole cell lysate, in the loading buffer containing double DTT at 40 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood), whole cell lysate, in the loading buffer containing double DTT at 40 µg

Lane 5:

SW480 (human colorectal adenocarcinoma epithelial cell), whole cell lysate, in the loading buffer containing double DTT at 40 µg

Lane 6:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate, in the loading buffer containing double DTT at 40 µg

Lane 7:

AR42J (rat pancreatic tumor epithelial cell), whole cell lysate, in the loading buffer containing double DTT at 40 µg

Lane 8:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole fresh cell lysate at 40 µg

Lane 9:

HepG2 (human hepatocelullar carcinoma epithelial cell) whole fresh cell lysate at 40 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 1/100000 dilution

Observed band size: 260 kDa,124 kDa

false

• Dot

Supplier Data

Dot Blot - Anti-ITPR2 antibody [EPR28319-63] (AB320725)

Dot blot analysis of ITPR2 using ab320725 at 1 : 1000 (0.531 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : His-tagged human ITRP2 fragment
Lane2 : His-tagged human ITRP1 fragment
Lane3 : His-tagged human ITRP3 fragment

Exposure time : 180 seconds.

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

This antibody does not cross-react with human ITRP1 and ITRP3.

Anti-His tag® antibody (1 : 10000) as total protein control.

All lanes:

Dot Blot - Anti-ITPR2 antibody [EPR28319-63] (ab320725) at 1/1000 dilution

Lane 1:

His-tagged human ITRP2 fragment

Lane 2:

His-tagged human ITRP1 fragment

Lane 3:

His-tagged human ITRP3 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