Anti-CD69 antibody [RM1274]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD69 antibody [RM1274] (AB322534)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labelling CD69 with ab322534 at 1/500 (0.98 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 cytoplasmic staining in human PBMC (shown in green) treated with ionomycin (500 ng/mL) and phorbol-12-myristate-13-acetate (10 ng/mL) for 3 hr. 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).

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.

• Flow Cyt

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Flow Cytometry - Anti-CD69 antibody [RM1274] (AB322534)

Flow cytometric analysis of Human PBMC untreated with 300ng/mL ionomycin and 10ng/mL PMA for 3 hours(up left and up right) / Human PBMC treated with 300ng/mL ionomycin and 10ng/mL PMA for 3 hours(down left and down right) cells labelling CD69 with ab322534 at 1/500 dilution (0.1ug) / down right compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

Human PBMC were stained with rabbit IgG or ab322534. Then stained with CD3 conjugated BV421 (450/50BP).
Gated on viable cell.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD69 antibody [RM1274] (AB322534)

Immunohistochemical analysis of paraffin-embedded Human gastric carcinoma tissue labeling CD69 with ab322534 at 1/500 (0.98 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human gastric carcinoma. The section was incubated with ab322534 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-CD69 antibody [RM1274] (AB322534)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD69 with ab322534 at 1/500 (0.98 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil. The section was incubated with ab322534 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-CD69 antibody [RM1274] (AB322534)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling CD69 with ab322534 at 1/500 (0.98 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on human skeletal muscle. The section was incubated with ab322534 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-CD69 antibody [RM1274] (AB322534)

CD69 was immunoprecipitated from 0.35 mg Daudi treated with 50ng/ml PMA for 24 hours, whole cell lysate with ab322534 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322534 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Daudi treated with 50ng/ml PMA for 24 hours, whole cell lysate
Lane 2 : ab322534 IP in Daudi treated with 50ng/ml PMA for 24 hours, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322534 Daudi treated with 50ng/ml PMA for 24 hours, whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-CD69 antibody [RM1274] (ab322534) at 1/30 dilution

All lanes:

Daudi treated with 50ng/ml PMA for 24 hours, whole cell lysate

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

false

Exposure time: 10s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD69 antibody [RM1274] (AB322534)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse PBMC (mouse peripheral blood mononuclear cell) cells labelling CD69 with ab322534 at 1/500 (0.98 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 cytoplasmic staining in mouse PBMC (shown in green) treated with ionomycin (500 ng/mL) and phorbol-12-myristate-13-acetate (10 ng/mL) for 24 hr. 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).

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.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD69 antibody [RM1274] (AB322534)

Flow cytometric analysis of Mouse PBMC untreated with 500ng/mL ionomycin and 10ng/mL PMA for 24 hours(up left and up right) / Mouse PBMC treated with 500ng/mL ionomycin and 10ng/mL PMA for 24 hours(down left and down right) cells labelling CD69 with ab322534 at 1/500 dilution (0.1ug) / down right compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

Mouse PBMC were stained with rabbit IgG or ab322534. Then stained with CD3 conjugated Alexa Fluor®647 (670/14BP).
Gated on viable cell.

• WB

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Western blot - Anti-CD69 antibody [RM1274] (AB322534)

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

The expression of CD69 is upregulated/downregulated in response to PMA treatment (PMID : 1617156).

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

All lanes:

Western blot - Anti-CD69 antibody [RM1274] (ab322534) at 1/1000 dilution

Lane 1:

Untreated Daudi (human Burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 2:

Daudi treated with 50ng/ml PMA for 24 hours, 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: 28-32 kDa,36 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-CD69 antibody [RM1274] (AB322534)

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

Negative control : HeLa, 293T.

Low expression : K-562, skeletal muscle.

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

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

All lanes:

Western blot - Anti-CD69 antibody [RM1274] (ab322534) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

U-937 (human histiocytic lymphoma monocyte) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 5:

H9 (human lymphoma cutaneous T lymphocyte) whole cell lysate at 20 µg

Lane 6:

Human tonsil tissue lysate at 20 µg

Lane 7:

Human skeletal muscle tissue lysate at 20 µg

Lane 8:

Human thymus tissue 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: 28-32 kDa,36 kDa

false

Exposure time: 125s