Anti-nNOS (neuronal) antibody [RM1237]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of paraffin-embedded Human cerebellum tissue labeling nNOS (neuronal) with ab323183 at 1/100 (5.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cerebellum. The section was incubated with ab323183 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-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling nNOS (neuronal) with ab323183 at 1/100 (5.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on myenteric nerve plexus of human colon. The section was incubated with ab323183 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-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling nNOS (neuronal) with ab323183 at 1/100 (5.05 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on human spleen. The section was incubated with ab323183 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-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a human NOS1 expression vector containing a myc-His-tag cell pellet. (B) HEK-293T transfected with a human NOS2 expression vector containing a myc-His-tag cell pellet. (C) HEK-293T transfected with a human NOS3 expression vector containing a myc-His-tag cell pellet. (D) HEK-293T transfected with an empty vector containing a His-tag. tissue labeling nNOS (neuronal) with ab323183 at 1/5000 (0.101 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) HEK-293T transfected with a human NOS1 expression vector containing a myc-His-tag cell pellet, no staining on (B) HEK-293T transfected with a human NOS2 expression vector containing a myc-His-tag cell pellet, (C) HEK-293T transfected with a human NOS3 expression vector containing a myc-His-tag cell pellet, and (D) HEK-293T transfected with an empty vector containing a His-tag cell pellet. The section was incubated with ab323183 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-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 4T1(mouse mammary gland carcinoma epithelial cell) cells labelling nNOS (neuronal) with ab323183 at 1/2000 (0.253 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green).

Low expression : Confocal image showing no staining in 4T1 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).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Magenta).

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural/glia cells labelling nNOS (neuronal) with ab323183 at 1/2000 (0.253 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green).

Confocal image showing cytoplasmic 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). 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).

ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 (4ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2ug/ml) dilution (Magenta).

-ve control 1 : ab323183 at a 1/2000 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : ab11267 at a 1/500 dilution followed by ab150081 at a 1/1000 dilution

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebellum (fresh frozen) tissue labeling nNOS (neuronal) with ab323183 at 1/100 (5.05 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-nNOS (neuronal) (ab323183, green), anti-Synapsin I (ab309972, magenta) and anti-NeuN (ab190565, ) on mouse cerebellum.
Panel B : anti-nNOS (neuronal) stained on mouse cerebellum.
Panel C : anti-Synapsin I stained in neurons of mouse cerebellum.
Panel D : anti-NeuN stained in neurons of mouse cerebellum.
The section was incubated in two rounds of staining : in the order of ab323183 and ab309972, ab190565 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.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebellum (fresh frozen) tissue labeling nNOS (neuronal) with ab323183 at 1/100 (5.05 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-nNOS (neuronal) (ab323183, green), anti-Synapsin I (ab309972, magenta) and anti-NeuN (ab190565, ) on rat cerebellum.
Panel B : anti-nNOS (neuronal) stained on rat cerebellum.
Panel C : anti-Synapsin I stained in neurons of rat cerebellum.
Panel D : anti-NeuN stained in neurons of rat cerebellum.
The section was incubated in two rounds of staining : in the order of ab323183 and ab309972, ab190565 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling nNOS (neuronal) with ab323183 at 1/50 (10.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on rat spleen. The section was incubated with ab323183 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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat primary neural/glia cells labelling nNOS (neuronal) with ab323183 at 1/2000 (0.253 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green).

Confocal image showing cytoplasmic 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). 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).

ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 (4ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2ug/ml) dilution (Magenta).

-ve control 1 : ab323183 at a 1/2000 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : ab11267 at a 1/500 dilution followed by ab150081 at a 1/1000 dilution

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling nNOS (neuronal) with ab323183 at 1/50 (10.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum. The section was incubated with ab323183 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-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling nNOS (neuronal) with ab323183 at 1/50 (10.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat cerebrum. The section was incubated with ab323183 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-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling nNOS (neuronal) with ab323183 at 1/50 (10.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on mouse spleen. The section was incubated with ab323183 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-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat spleen (fresh frozen) tissue labeling nNOS (neuronal) with ab323183 at 1/100 (5.05 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 ab323183 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-nNOS (neuronal) antibody [RM1237] (AB323183)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen (fresh frozen) tissue labeling nNOS (neuronal) with ab323183 at 1/100 (5.05 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 ab323183 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.

• WB

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Western blot - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

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

Negative control : HeLa

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

All lanes:

Western blot - Anti-nNOS (neuronal) antibody [RM1237] (ab323183) at 1/1000 dilution

Lane 1:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma 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 1/100000 dilution

Observed band size: 161 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

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

Low expression tissue : spleen, pancreas

The identity of the lower MW band at approximately 100 kDa (in lane 1) is unknown.

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution (15 kDa).

All lanes:

Western blot - Anti-nNOS (neuronal) antibody [RM1237] (ab323183) at 1/1000 dilution

Lane 1:

Human hippocampus tissue lysate at 20 µg

Lane 2:

Human spleen tissue lysate at 20 µg

Lane 3:

Human pancreas 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: 161 kDa,15 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

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

Low expression : 4T1

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

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

All lanes:

Western blot - Anti-nNOS (neuronal) antibody [RM1237] (ab323183) at 1/1000 dilution

Lane 1:

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

Lane 2:

4T1 (mouse mammary gland carcinoma 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 1/100000 dilution

Observed band size: 161 kDa,124 kDa

true

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-nNOS (neuronal) antibody [RM1237] (AB323183)

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

Low expression tissue : spleen

The identity of the lower MW band at approximately 100 kDa (in lane 1, 2, 4, 5) is unknown.

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

All lanes:

Western blot - Anti-nNOS (neuronal) antibody [RM1237] (ab323183) at 1/1000 dilution

Lane 1:

Mouse cerebellum tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Mouse spleen tissue lysate at 20 µg

Lane 4:

Rat cerebellum tissue lysate at 20 µg

Lane 5:

Rat brain tissue lysate at 20 µg

Lane 6:

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: 161 kDa,36 kDa

false

Exposure time: 10s