Anti-HHLA2 antibody [RM2075]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HHLA2 antibody [RM2075] (AB323612)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling HHLA2 with ab323612 at 1/500 (1.01 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon carcinoma. The section was incubated with ab323612 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HHLA2 antibody [RM2075] (AB323612)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling HHLA2 with ab323612 at 1/500 (1.01 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human spleen. The section was incubated with ab323612 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

• Flow Cyt

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Flow Cytometry - Anti-HHLA2 antibody [RM2075] (AB323612)

Flow cytometric analysis of Jurkat (human T cell leukemia T lymphocyte from peripheral blood, Left) /A-204 (human muscle rhabdomyosarcoma cell, Right) cells labelling HHLA2 with ab323612 at 1/5000 dilution (0.01 ug) / Right 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® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Negative control : Jurkat (PMID : 25549724).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HHLA2 antibody [RM2075] (AB323612)

Immunohistochemical analysis of paraffin-embedded (A) A-204 (human muscle rhabdomyosarcoma cell) cell pellet (B) HeLa (human cervical adenocarcinoma epithelial cell) cell pellet labeling HHLA2 with ab323612 at 1/500 (1.01 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on A-204 (image A). No staining on HeLa (image B). The section was incubated with ab323612 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

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Immunocytochemistry/ Immunofluorescence - Anti-HHLA2 antibody [RM2075] (AB323612)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized A-204 (human muscle rhabdomyosarcoma cell) cells labelling HHLA2 with ab323612 at 1/50 (10.1 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 mainly membranous staining in A-204 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : Jurkat (PMID : 25549724).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HHLA2 antibody [RM2075] (AB323612)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling HHLA2 with ab323612 at 1/500 (1.01 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon. The section was incubated with ab323612 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-HHLA2 antibody [RM2075] (AB323612)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a human HHLA2 expression vector containing a Myc-His tag. (B) HEK-293T transfected with empty vector containing a Myc-His tag. tissue labeling HHLA2 with ab323612 at 1/2000 (0.253 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on HEK-293T transfected with a Myc-His-tagged human HHLA2 construct (image A). No staining on HEK-293T transfected with empty plasmid (image B). The section was incubated with ab323612 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

• IP

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Immunoprecipitation - Anti-HHLA2 antibody [RM2075] (AB323612)

HHLA2 was immunoprecipitated from 0.35 mg A-204 (human muscle rhabdomyosarcoma cell) whole cell lysate with ab323612 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab323612 at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-HHLA2 antibody [RM2075] (ab323612) at 1/1000 dilution

Lane 1:

(Input) A-204 (human muscle rhabdomyosarcoma cell) whole cell lysate at 10 µg

Lane 2:

ab323612 at 1/30 IP in A-204 (human muscle rhabdomyosarcoma cell) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab323612 in A-204 whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 70-80 kDa

false

Exposure time: 6s

• WB

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Western blot - Anti-HHLA2 antibody [RM2075] (AB323612)

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

Negative control : HeLa, Jurkat (PMID : 25549724), HepG2.

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

Exposure time : Lanes 1-3 : 26 seconds; Lanes 4-7 : 6 seconds.

All lanes:

Western blot - Anti-HHLA2 antibody [RM2075] (ab323612) at 1/1000 dilution

Lane 1:

AsPC-1 (human Pancreas epithelial cell) whole cell lysate at 20 µg

Lane 2:

HCC827 (human lung adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HDLM-2 (human hodgkin lymphoma cell) whole cell lysate at 20 µg

Lane 4:

A-204 (human muscle rhabdomyosarcoma cell) whole cell lysate at 20 µg

Lane 5:

Jurkat (human t cell leukemia t lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 6:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 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: 70-80 kDa,36 kDa

false