Anti-Annexin V/ANXA5 antibody [RM1317]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling Annexin V/ANXA5 with ab325388 at 1/20000 (0.026 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human placenta. 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).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Annexin V/ANXA5 with ab325388 at 1/20000 (0.026 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon. 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).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Annexin V/ANXA5 with ab325388 at 1/20000 (0.026 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

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).

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell) (Green and black) and ANXA5 KO HAP1(HO010326) (Magenta and grey) cells labelling Annexin V/ANXA5 with ab325388 at 1/500 dilution (0.1ug) / Green and magenta compared with a Rabbit monoclonal IgG (ab172730) / Black and Grey isotype control.

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized ANXA5 KO HAP1(ANXA5 knockout human chronic myelogenous leukemia near-haploid cell); Wildtype HAP 1 (human chronic myelogenous leukemia near-haploid cell) cells labelling Annexin V/ANXA5 with ab325388 at 1/2000 (0.259 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing mainly membranous and weak nuclear, cytoplasmic staining in wildtype HAP1 cells (shown in green), showing no staining in ANXA5 knockout HAP1 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).

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

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) cell pellet and (B) ANXA5 knockout HAP1 cell pellet labeling Annexin V/ANXA5 with ab325388 at 1/10000 (0.052 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) cell pellet (Image A) and no staining on ANXA5 knockout HAP1 cell pellet (Image B).

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).

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

• IP

Lab

Immunoprecipitation - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Annexin V/ANXA5 was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) whole cell lysate with ab325388 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325388 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : ab325388 at 1/30 IP in 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325388 in 293T whole cell lysate

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-Annexin V/ANXA5 antibody [RM1317] (ab325388) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab325388 at 1/30 IP in 293T (human embryonic kidney epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325388 in 293Twhole 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

Observed band size: 35 kDa

false

Exposure time: 10s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling Annexin V/ANXA5 with ab325388 at 1/10000 (0.052 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat colon. 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).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling Annexin V/ANXA5 with ab325388 at 1/10000 (0.052 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat 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).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling Annexin V/ANXA5 with ab325388 at 1/20000 (0.052 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse 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).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunohistochemical analysis of paraffin-embedded (A) Colon tissue from wild-type C57BL/6J mice and (B) Colon tissue from ANXA5 knockout mice tissue labeling Annexin V/ANXA5 with ab325388 at 1/20000 (0.026 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) colon tissue from wild-type C57BL/6J mice and no staining on (B) colon tissue from ANXA5 knockout mice.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Anxa5-KO homozygous mice (Strain ID : T028565)

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

Immunohistochemical analysis of paraffin-embedded (A) Liver tissue from wild-type C57BL/6J mice and (B) Liver tissue from ANXA5 knockout mice tissue labeling Annexin V/ANXA5 with ab325388 at 1/20000 (0.026 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) liver tissue from wild-type C57BL/6J mice and no staining on (B) liver tissue from ANXA5 knockout mice.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Anxa5-KO homozygous mice (Strain ID : T028565)

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

Lab

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

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

Performed under reducing conditions.

In Western blot, ab325388 was shown to bind specifically to ANXA5. Target of interest was observed at 35 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in ANXA5 knockout cell line (lane 2).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (ab325388) at 1/5000 dilution

Lane 1:

Wild-type HAP1(human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 10 µg

Lane 2:

ANXA5 knockout HAP1 whole cell lysate at 10 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 4:

293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 5:

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

Lane 6:

C2C12 (mouse myoblast) whole cell lysate at 10 µg

Lane 7:

C6 (rat glial tumor glial cell) whole cell lysate at 10 µg

Lane 8:

PC-12 (rat adrenal gland pheochromocytoma cell) 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

Observed band size: 35 kDa,36 kDa

false

Exposure time: 59s

• WB

Lab

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

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

All lanes:

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (ab325388) at 1/5000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Human liver tissue lysate at 20 µg

Lane 3:

Human testis tissue lysate at 20 µg

Lane 4:

Human spleen 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 1/100000 dilution

Observed band size: 35 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

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

The tissue samples in lane 3-4 were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and ANXA5-KO homozygous mice (Strain ID : T028565).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (ab325388) at 1/1000 dilution

Lane 1:

Rat colon tissue lysate at 20 µg

Lane 2:

Rat liver tissue lysate at 20 µg

Lane 3:

Wild-type mouse liver tissue lysate at 10 µg

Lane 4:

ANXA5 knockout mouse liver tissue 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

Observed band size: 35 kDa,36 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

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

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and ANXA5-KO homozygous mice (Strain ID : T028565)

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002)(1/5000)(124KDa).

All lanes:

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (ab325388) at 1/5000 dilution

Lane 1:

Wild-type mouse heart tissue lysate at 10 µg

Lane 2:

ANXA5 knockout mouse heart tissue lysate at 10 µg

Lane 3:

Wild-type mouse colon tissue lysate at 10 µg

Lane 4:

ANXA5 knockout mouse colon tissue lysate at 10 µg

Lane 5:

Wild-type mouse kidney tissue lysate at 10 µg

Lane 6:

ANXA5 knockout mouse kidney tissue 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

Observed band size: 35 kDa,124 kDa

false

Exposure time: 59s

• WB

Lab

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (AB325388)

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

All lanes:

Western blot - Anti-Annexin V/ANXA5 antibody [RM1317] (ab325388) at 1/5000 dilution

Lane 1:

Rat brain tissue lysate at 20 µg

Lane 2:

Rat heart tissue lysate at 20 µg

Lane 3:

Rat kidney tissue lysate at 20 µg

Lane 4:

Rat spleen 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 1/100000 dilution

Observed band size: 35 kDa

false

Exposure time: 15s