Anti-Annexin A1/ANXA1 antibody [EPR19342]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic staining on human tonsil tissue is observed [PMID : 9720986].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Immunohistochemical analysis of paraffin-embedded human endometrial cancer tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic and weak membrane staining on human endometrial cancer tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized BxPC-3 (Human pancreas adenocarcinoma cell line) cells labeling Annexin A1 with ab214486 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membrane and weak cytoplasmic staining on BxPC-3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic staining on human breast tissue is observed [PMID : 16949910].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear, cytoplasmic and membrane staining on human bladder cancer tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

ab214486 staining ANXA1 in HeLa cells. The cells were fixed with 4% paraformaldehyde (10min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab214486 at 5ug/ml concentration and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Annexin A1 with ab214486 at 1/600 dilution (red) compared withRabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Annexin A1 with ab214486 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing membrane and cytoplasmic staining on NIH/3T3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Immunohistochemical analysis of paraffin-embedded rat lung tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic staining on rat lung tissue is observed [PMID : 15133855] [PMID : 9720986].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling Annexin A1 with ab214486 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and cytoplasmic staining on mouse spleen tissue is observed [PMID : 9720986].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

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Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 3 minutes; Lane 2 : 15 seconds; Lane 3 : 1 second.

Lanes 1 - 2:

Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/5000 dilution

Lane 3:

Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/20000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Lane 3:

Human placenta lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 38 kDa

Observed band size: 33 kDa,37 kDa

false

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

ab214486 staining ANXA1 in wild-type Hap1 cells (top panel) and ANXA1 knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab214486 at 5ug/ml concentration and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IP

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Immunoprecipitation - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Annexin A1 was immunoprecipitated from 0.35 mg of K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate with ab214486 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab214486 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

Lane 1 : K562 whole cell lysate 10μg (Input).

Lane 2 : ab214486 IP in K562 whole cell lysate.

Lane 3 : Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab214486 in K562 whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486)

Predicted band size: 38 kDa

false

• WB

Supplier Data

Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Blocking/Dilution buffer : 5% NFDM/TBST.

The two band 37kDa is the full length band and the 33kDa supposed to be the cleavage form, this expression pattern observed is consistent with what has been described in the literature (PMID : 25510623 and 20679535).

All lanes:

Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/10000 dilution

Lane 1:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 10 µg

Lane 2:

BxPC-3 (Human pancreas adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 3:

C2C12 (Mouse myoblast cell line) whole cell lysate at 10 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

Predicted band size: 38 kDa

Observed band size: 33 kDa,37 kDa

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 3 minutes; Lane 2/6/7 : 1 seconds; Lane 3 : 15 seconds; Lane 4/5 : 30 seconds.

Lanes 1 - 2:

Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/5000 dilution

Lanes 3 - 7:

Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486) at 1/2000 dilution

Lane 1:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Lane 4:

Mouse kidney lysate at 10 µg

Lane 5:

Mouse spleen lysate at 10 µg

Lane 6:

Rat kidney lysate at 10 µg

Lane 7:

Rat spleen lysate at 10 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

Predicted band size: 38 kDa

Observed band size: 33 kDa,37 kDa

false

• WB

Lab

Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (AB214486)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : Annexin A1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab214486 observed at 38 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab214486 was shown to specifically react with Annexin A1 when Annexin A1 knockout samples were used. Wild-type and Annexin A1 knockout samples were subjected to SDS-PAGE. ab214486 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Annexin A1/ANXA1 antibody [EPR19342] (ab214486)

Predicted band size: 38 kDa

false