Anti-nNOS (phospho S847) antibody [EPR28208-546]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

Immunohistochemical analysis of paraffin-embedded (A) Mouse cerebrum without alkaline phosphatase treatment and (B) Mouse cerebrum with alkaline phosphatase treatment tissue labeling nNOS (neuronal) with ab325748 at 1/500 (1.008 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) mouse cerebrum without alkaline phosphatase treatment, no staining on (B) mouse cerebrum with alkaline phosphatase treatment. 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 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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebrum (perfused fixed) tissue labeling nNOS (neuronal) with ab325748 at 1/50 (10.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing positive staining on rat cerebrum, and the signal decreased after phosphatase treatment at 37℃ for 2 hr. Panel A, a : merged staining of anti-NOS1 pS847 (ab325748, green), anti-NeuN (ab190565, magenta) on rat cerebrum. Panel B, b : anti-NOS1 pS847 stained on rat cerebrum. Panel C, c : anti-NeuN stained in neurons of rat cerebrum. The section was incubated in two rounds of staining : in the order of ab325748 and 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.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

Immunohistochemical analysis of paraffin-embedded (A) Rat cerebrum without alkaline phosphatase treatment and (B) Rat cerebrum with alkaline phosphatase treatment tissue labeling nNOS (neuronal) with ab325748 at 1/500 (1.008 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) rat cerebrum without alkaline phosphatase treatment, no staining on (B) ratcerebrum with alkaline phosphatase treatment. 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 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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (perfused fixed) tissue labeling nNOS (neuronal) with ab325748 at 1/50 (10.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing positive staining on mouse cerebrum, and the signal decreased after phosphatase treatment at 37℃ for 2 hr. Panel A, a : merged staining of anti-NOS1 pS847 (ab325748, green), anti-NeuN (ab190565, magenta) on mouse cerebrum. Panel B, b : anti-NOS1 pS847 stained on mouse cerebrum. Panel C, c : anti-NeuN stained in neurons of mouse cerebrum. The section was incubated in two rounds of staining : in the order of ab325748 and 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.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

• WB

Lab

Western blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

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

All lanes:

Western blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (ab325748) at 1/1000 dilution

Lane 1:

Mouse cerebellum tissue lysate (untreated membrane) at 20 µg

Lane 2:

Mouse cerebellum tissue lysate (alkaline phosphatase treated membrane) 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: 160 kDa

false

Exposure time: 103s

• WB

Lab

Western blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

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

All lanes:

Western blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (ab325748) at 1/1000 dilution

Lane 1:

Rat cerebellum tissue lysate (untreated membrane) at 20 µg

Lane 2:

Rat cerebellum tissue lysate (alkaline phosphatase treated membrane) 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: 160 kDa

false

Exposure time: 81s

• WB

Lab

Western blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

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

Negative control : liver (PMID : 8626668).

Low expression : spleen (PMID : 8626668).

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

All lanes:

Western blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (ab325748) at 1/1000 dilution

Lane 1:

Mouse cerebellum tissue lysate at 20 µg

Lane 2:

Mouse spinal cord tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Mouse spleen tissue lysate at 20 µg

Lane 5:

Rat cerebellum tissue lysate at 20 µg

Lane 6:

Rat spinal cord tissue lysate at 20 µg

Lane 7:

Rat liver tissue lysate at 20 µg

Lane 8:

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: 160 kDa,124 kDa

false

Exposure time: 70s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling nNOS (neuronal) with ab325748 at 1/500 (1.008 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control : no staining on mouse liver. 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 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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (perfused fixed) tissue labeling nNOS (neuronal) with ab325748 at 1/50 (10.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Negative control : confocal image showing no staining on mouse liver. The nuclear counterstain was DAPI (Blue). The section was incubated with ab325748 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.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling nNOS (neuronal) with ab325748 at 1/500 (1.008 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control : no staining on rat liver. 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 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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat liver (perfused fixed) tissue labeling nNOS (neuronal) with ab325748 at 1/50 (10.08 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Negative control : confocal image showing no staining on rat liver. The nuclear counterstain was DAPI (Blue). The section was incubated with ab325748 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.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

• Dot

Lab

Dot Blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

Dot blot analysis of nNOS (neuronal) using ab325748 at 1 : 1000 (0.504 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane 1 : Mouse nNOS (phospho S847) peptide

Lane 2 : Mouse nNOS non-phospho peptide

Exposure time : 180 seconds.

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

All lanes:

Dot Blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (ab325748) at 1/1000 dilution

Lane 1:

Mouse nNOS (phospho S847) peptide

Lane 2:

Mouse nNOS non-phospho peptide

Secondary

All lanes:

Dot 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

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (AB325748)

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

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000) (160kDa).

All lanes:

Western blot - Anti-nNOS (phospho S847) antibody [EPR28208-546] (ab325748) at 1/5000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with mouse wild-type nNOS expression vector containing a myc-His-tag® whole cell lysate (untreated membrane) at 2 µg

Lane 2:

293T cells transfected with mouse nNOS (mutation S847A) expression vector containing a myc-His-tag® whole cell lysate (untreated membrane) at 2 µg

Lane 3:

293T cells transfected with mouse wild-type nNOS expression vector containing a myc-His-tag® whole cell lysate (alkaline phosphatase treated membrane) at 2 µg

Lane 4:

293T cells transfected with mouse nNOS (mutation S847A) expression vector containing a myc-His-tag® whole cell lysate (alkaline phosphatase treated membrane) at 2 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: 160 kDa

false

Exposure time: 8s