Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Immunohistochemical analysis of paraffin-embedded Rat retina tissue labeling BRN3A + BRN3B + BRN3C with ab317492 at 1/1000 (0.535 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on rat retina. The section was incubated with ab317492 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized mouse primary neural/glia cells labeling BRN3A + BRN3B + BRN3C with ab317492 at 1/200 dilution (2.575 μg/ml), followed by ab150081 AlexaFluor®488 Goat anti-Rabbit secondary preadsorbed at 1/1000 dilution (2 μg/ml) (Green). Confocal image showing nuclear staining in mouse primary neurons (shown in green). Counterstained with ab11267 anti-MAP2 (mouse mAb) at 1/1000 dilution (1 μg/ml), followed by ab150120 AlexaFluor®594 Goat anti-Mouse secondary at 1/1000 dilution (2 μg/ml) (Magenta). Nuclear DNA was labelled with DAPI (shown in blue). Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). -ve control 1 : ab317492 at a 1/200 dilution (2.575 μg/ml), followed by ab150120 at a 1/1000 dilution (2 μg/ml). -ve control 2 : ab11267 at a 1/500 dilution (4 μg/ml), followed by ab150081 at a 1/1000 dilution (2 μg/ml).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized rat primary neural/glia cells labeling BRN3A + BRN3B + BRN3C with ab317492 at 1/200 dilution (2.575 μg/ml), followed by ab150081 AlexaFluor®488 Goat anti-Rabbit secondary preadsorbed at 1/1000 dilution (2 μg/ml) (Green). Confocal image showing nuclear staining in rat primary neurons (shown in green). Counterstained with ab11267 anti-MAP2 (mouse mAb) at 1/1000 dilution (1 μg/ml), followed by ab150120 AlexaFluor®594 Goat anti-Mouse secondary at 1/1000 dilution (2 μg/ml) (Magenta). Nuclear DNA was labelled with DAPI (shown in blue). Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). -ve control 1 : ab317492 at a 1/200 dilution (2.575 μg/ml), followed by ab150120 at a 1/1000 dilution (2 μg/ml). -ve control 2 : ab11267 at a 1/500 dilution (4 μg/ml), followed by ab150081 at a 1/1000 dilution (2 μg/ml).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Immunohistochemical analysis of paraffin-embedded Mouse retina tissue labeling BRN3A + BRN3B + BRN3C with ab317492 at 1/1000 (0.535 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on mouse retina. The section was incubated with ab317492 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Immunohistochemical analysis of paraffin-embedded Mouse E14.5 tissue labeling BRN3A + BRN3B + BRN3C with ab317492 at 1/1000 (0.535 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on mouse E14.5. The section was incubated with ab317492 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling BRN3A + BRN3B + BRN3C with ab317492 at 1/1000 (0.535 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control : no staining on mouse cardiac muscle. The section was incubated with ab317492 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Immunohistochemical analysis of paraffin-embedded Rat cardiac muscle tissue labeling BRN3A + BRN3B + BRN3C with ab317492 at 1/1000 (0.535 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control : no staining on rat cardiac muscle. The section was incubated with ab317492 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized mouse splenocytes labeling BRN3A + BRN3B + BRN3C with ab317492 at 1/200 dilution (2.575 μg/ml), followed by ab150081 AlexaFluor®488 Goat anti-Rabbit secondary preadsorbed at 1/1000 dilution (2 μg/ml) (Green). Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at a 1/200 dilution (2.5 μg/ml) (magenta). Confocal image showing negative staining in mouse splenocytes. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). Negative control：splenocyte (PMID : 11470235).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized rat splenocytes labeling BRN3A + BRN3B + BRN3C with ab317492 at 1/200 dilution (2.575 μg/ml), followed by ab150081 AlexaFluor®488 Goat anti-Rabbit secondary preadsorbed at 1/1000 dilution (2 μg/ml) (Green). Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at a 1/200 dilution (2.5 μg/ml) (magenta). Confocal image showing negative staining in rat splenocytes. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). Negative control：splenocyte (PMID : 11470235).

• IP

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Immunoprecipitation - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

BRN3A + BRN3B + BRN3C was immunoprecipitated from 0.35 mg Rat E15 brain tissue lysate with ab317492 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317492 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Rat E15 brain tissue lysate
Lane 2 : ab317492 IP in Rat E15 brain tissue lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317492 in rat E15 brain tissue lysate

All lanes:

Immunoprecipitation - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (ab317492) at 1/30 dilution

All lanes:

Rat E15 brain tissue 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: 180s

• WB

Supplier Data

Western blot - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

The lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

The identity of the bands lower than 30 kDa are unknown

All lanes:

Western blot - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (ab317492) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast) 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 1/100000 dilution

Observed band size: 45 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

Negative control : kidney (PMID : 1357630, 11470235)

The identity of the bands lower than 30 kDa in lane 2 and lane 3 are unknown.

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

Exposure time : Lanes 1-2 : 180 seconds; Lane 3 : 92 seconds.

All lanes:

Western blot - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (ab317492) at 1/1000 dilution

Lane 1:

Rat E15 brain tissue lysate at 20 µg

Lane 2:

Rat kidney tissue lysate at 20 µg

Lane 3:

Mouse E14.5 brain tissue lysate at 48 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: 45 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (AB317492)

This antibody recognizes BRN3A, BRN3B and BRN3C.

This antibody does not recognize POU3F2/Brn-2, POU3F3/Brain1, POU3F4/BRN4 and POU3F1/Oct6.

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-BRN3A + BRN3B + BRN3C antibody [EPR26313-54] (ab317492) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 2:

293T transfected with a human BRN3A protein expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 3:

293T transfected with a human BRN3B protein expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 4:

293T transfected with a human BRN3C protein expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 5:

293T transfected with a human POU3F2/Brn-2 protein expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 6:

293T transfected with a human POU3F3/Brain1 protein expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 7:

293T transfected with a human POU3F4/BRN4 protein expression vector containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 8:

293T transfected with a human POU3F1/Oct6 protein expression vector containing a myc-His-tag®, 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: 20-65 kDa,15 kDa

false

Exposure time: 3s