Anti-Neogenin antibody [EPR30444-522]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of paraffin-embedded A Wild-type A549 (human lung carcinoma epithelial cell) cell pellet B NEO1 knockout A549 cell pellet tissue labeling Neogenin with ab324107 at 1/1000 (0.488 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on A : wild-type A549 cell pellet, no staining on B : NEO1 knockout A549 cell pellet
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-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Neogenin with ab324107 at 1/1000 (0.488 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-Neogenin antibody [EPR30444-522] (AB324107)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse splenocyte cells labelling Neogenin with ab324107 at 1/50 (9.76 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Low expression : confocal image showing no staining in mouse splenocytes (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).

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 : ab324107 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) antibody at 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat splenocyte cells labelling Neogenin with ab324107 at 1/50 (9.76 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Low expression : confocal image showing no staining in rat splenocytes (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).

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 : ab324107 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) antibody at 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural/glia cell cells labelling Neogenin with ab324107 at 1/50 (9.76 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing positive staining in mouse primary neural/glia cells (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 : ab324107 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) antibody at 1/1000 dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse hippocampus (fresh frozen) tissue labeling Neogenin with ab324107 at 1/50 (9.76 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-Neogenin (ab324107, green), anti-NeuN (ab190565, white) and anti-GFAP (ab201732, magenta) on mouse hippocampus.
Panel B : anti-Neogenin 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 ab324107 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).

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat spleen (fresh frozen) tissue labeling Neogenin with ab324107 at 1/50 (9.76 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 rat spleen. The nuclear counterstain was DAPI (Blue). The section was incubated with ab324107 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) preadsorbedat 1/1000 2 ug/mL dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen (fresh frozen) tissue labeling Neogenin with ab324107 at 1/50 (9.76 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 spleen. The nuclear counterstain was DAPI (Blue). The section was incubated with ab324107 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) preadsorbedat 1/1000 2 ug/mL dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat primary neural/glia cell cells labelling Neogenin with ab324107 at 1/50 (9.76 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing positive staining in rat primary neural/glia cells (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 : ab324107 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) antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of paraffin-embedded A Neuro-2a (mouse neuroblastoma neuroblast) cell pellet B A20 (mouse reticulum sarcoma B lymphocyte) cell pellet tissue labeling Neogenin with ab324107 at 1/1000 (0.488 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on A : Neuro-2a cell pellet, no staining on B : A20 cell pellet.
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-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling Neogenin with ab324107 at 1/1000 (0.488 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat 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

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat hippocampus (fresh frozen) tissue labeling Neogenin with ab324107 at 1/50 (9.76 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-Neogenin (ab324107, green), anti-NeuN (ab190565, white) and anti-GFAP (ab201732, magenta) on rat hippocampus.
Panel B : anti-Neogenin stained on rat hippocampus.
Panel C : anti-NeuN stained in neurons of rat hippocampus.
Panel D : anti-GFAP stained in astrocytes of rat hippocampus.
The section was incubated in two rounds of staining : in the order of ab324107 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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling Neogenin with ab324107 at 1/1000 (0.488 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : almost no staining on rat spleen. 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-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling Neogenin with ab324107 at 1/1000 (0.488 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum (PMID : 16982849). 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

• WB

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Western blot - Anti-Neogenin antibody [EPR30444-522] (AB324107)

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

In Western blot, ab324107 was shown to bind specifically to Neogenin. Target of interest was observed at 140-200 kDa in wild-type A549 cell lysates (lane 8) with no signal observed at this size in NEO1 knockout cell line (lane 8) (lane 8, knockout cell line ab282825).

Low expression : A20, P815.

Samples are non-boiled as boiling may cause protein aggregation.

Lanes 3-8 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 and lanes 1-2 are incubated with Goat Anti-Rabbit IgG (HRP) at 1/2000.

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

Exposure time : Lanes 1-6 : 15 seconds, lanes 7-8 : 37 seconds

All lanes:

Western blot - Anti-Neogenin antibody [EPR30444-522] (ab324107) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Human hypothalamus tissue lysate at 20 µg

Lane 3:

4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 5:

A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate at 20 µg

Lane 6:

P815 (mouse mastocytoma mast Cell) whole cell lysate at 20 µg

Lane 7:

Wild-type A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 8:

NEO1 knockout A549 whole cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Lanes 3 - 8:

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: 140-200 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-Neogenin antibody [EPR30444-522] (AB324107)

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

Neogenin is a glycoprotein of approximately 200 kDa and detected as a 180-kDa band after treated with Peptide : N-glycosidase F (PNGase F).

Samples are non-boiled as boiling may cause protein aggregation.

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

All lanes:

Western blot - Anti-Neogenin antibody [EPR30444-522] (ab324107) at 1/1000 dilution

Lane 1:

Untreated Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate treated with Peptide: N-glycosidase F (PNGase F) 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: 180-200 kDa,124 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Neogenin antibody [EPR30444-522] (AB324107)

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

Low expression : spleen (PMID : 22412855).

Samples are non-boiled as boiling may cause protein aggregation.

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

All lanes:

Western blot - Anti-Neogenin antibody [EPR30444-522] (ab324107) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse liver tissue lysate at 20 µg

Lane 3:

Mouse testis tissue lysate at 20 µg

Lane 4:

Mouse 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 1/20000 dilution

Observed band size: 140-200 kDa,124 kDa

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-Neogenin antibody [EPR30444-522] (AB324107)

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

Low expression : spleen (PMID : 22412855).

Samples are non-boiled as boiling may cause protein aggregation.

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

All lanes:

Western blot - Anti-Neogenin antibody [EPR30444-522] (ab324107) at 1/1000 dilution

Lane 1:

Rat testis tissue lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Lane 3:

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 1/20000 dilution

Observed band size: 140-200 kDa,124 kDa

false

Exposure time: 26s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Neogenin with ab324107 at 1/1000 (0.488 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on mouse spleen.
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-Neogenin antibody [EPR30444-522] (AB324107)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling Neogenin with ab324107 at 1/1000 (0.488 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on human spleen.
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