Anti-Neurobeachin antibody [EPR30440-555]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunohistochemical analysis of paraffin-embedded Human brain (hippocampus section) tissue labeling Neurobeachin with ab324953 at 1/100 (5.0 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human brain. (A) Low-powered (magnification, x80) and (B) high-powered (magnification, x200) microscopic images.
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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunohistochemical analysis of paraffin-embedded Human lung tissue labeling Neurobeachin with ab324953 at 1/100 (5.0 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining on human lung (PMID : 20071347).
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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Neurobeachin with ab324953 at 1/100 (5.0 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cerebrum.
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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized IMR-32 cells labelling Neurobeachin with ab324953 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in IMR-32 cell line (shown in green). 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).
Negative control : T84

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab324953 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse splenocyte cells labelling Neurobeachin with ab324953 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Negative control : confocal image showing no staining in mouse splenocyte (shown in green). 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).

Anti-mouse CD3 rat monoclonal antibody (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 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 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunohistochemical analysis of paraffin-embedded Mouse brain (hippocampus section) tissue labeling Neurobeachin with ab324953 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse brain. (A) Low-powered (magnification, x20) and (B) high-powered (magnification, x200) microscopic images. (PMID : 11102458).
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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neuron cells labelling Neurobeachin with ab324953 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in mouse primary neuron (shown in green). 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).

ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab324953 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.

-ve control 2 : ab11267 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling Neurobeachin with ab324953 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum (PMID : 11102458).
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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Neurobeachin with ab324953 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : No staining on mouse lung.
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-Fr

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Immunohistochemistry (Frozen sections) - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse lung (fresh frozen) tissue labeling Neurobeachin with ab324953 at 1/500 (1.0 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Low expression : confocal image showing no staining on mouse lung. The nuclear counterstain was DAPI (Blue). The section was incubated with ab324953 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse hippocampus (fresh frozen) tissue labeling Neurobeachin with ab324953 at 1/500 (1.0 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Panel A : merged staining of anti-NBEA (ab324953, green), anti-NeuN (ab190565, white) and anti-GFAP (ab201732, magenta) on mouse hippocampus.
Panel B : anti-NBEA stained on mouse hippocampus.
Panel C : anti-NeuN stained in neurons of mouse hippocampus.
Panel D : anti-GFAP stained in astrocytes of mouse hippocampus.
The section was incubated in two rounds of staining : in the order of ab324953 and ab190565, ab201732 for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• WB

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Western blot - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

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

Negative control : T-84, JAR

In lanes 1-4, To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible. In lanes 5-8, the lysates were stored at -80°C prior to Western Blotting.

The bands beneath the target bands(327 kDa) are expected to be degradation products.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

Exposure time : Lanes 1-2 : 158 seconds; Lanes 3-8 : 180 seconds

All lanes:

Western blot - Anti-Neurobeachin antibody [EPR30440-555] (ab324953) at 1/1000 dilution

Lane 1:

IMR-32 (human neuroblastoma neuroblast) fresh whole cell lysate at 20 µg

Lane 2:

T84 (human colon epithelial cell) fresh whole cell lysate at 20 µg

Lane 3:

Y79 (human retinoblastoma cell) fresh whole cell lysate at 20 µg

Lane 4:

JAR (human placenta choriocarcinoma epithelial cell) fresh whole cell lysate at 20 µg

Lane 5:

Y79 (human retinoblastoma cell) whole cell lysate at 20 µg

Lane 6:

IMR-32 (human neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 7:

T84 (human colon epithelial cell) whole cell lysate at 20 µg

Lane 8:

JAR (human placenta choriocarcinoma 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: 327 kDa,130-327 kDa,124 kDa

false

• WB

Supplier Data

Western blot - Anti-Neurobeachin antibody [EPR30440-555] (AB324953)

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

Low expression : lung, spleen(PMID : 11102458)

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 11102458).

The bands beneath the target bands(327 kDa) are expected to be degradation products.

Lanes 1-3 of this blot were developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

To minimize protein degradation, tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-Neurobeachin antibody [EPR30440-555] (ab324953) at 1/1000 dilution

Lane 1:

Mouse brain fresh tissue lysate at 20 µg

Lane 2:

Mouse liver fresh tissue lysate at 20 µg

Lane 3:

Mouse spleen fresh 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: 130-327 kDa,124 kDa

true

Exposure time: 158s