Anti-eNOS antibody [RM1181] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human endometrioid carcinoma tissue labeling eNOS with ab317582 at 1/1000 (0.516 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on endothelial cells in human endometrioid carcinoma. The section was incubated with ab317582 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-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling eNOS with ab317582 at 1/1000 (0.516 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human placenta. The section was incubated with ab317582 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-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling eNOS with ab317582 at 1/1000 (0.516 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on endothelial cells of human spleen. The section was incubated with ab317582 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-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling eNOS with ab317582 at 1/1000 (0.516 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : almost no staining on human skeletal muscle. The section was incubated with ab317582 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

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach labelling eNOS with ab317582 at 1/500 (B) and CD34 with ab315802 at 1/2000 dilution (C). Opal Polymer HRP Ms + Rb was used as a secondary antibody. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-eNOS (magenta; Opal™520), anti-CD34 (green; Opal™570) on human stomach.
Panel B : anti-eNOS staining endothelium in human stomach.
Panel C : anti-CD34 staining endothelium in human stomach.
Panel D : Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab317582 and ab315802 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human endometrium labelling eNOS with ab317582 at 1/500 (B) and CD34 with ab315802 at 1/2000 dilution (C). Opal Polymer HRP Ms + Rb was used as a secondary antibody. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-eNOS (magenta; Opal™520), anti-CD34 (green; Opal™570) on human endometrium.
Panel B : anti-eNOS staining endothelium in human endometrium.
Panel C : anti-CD34 staining endothelium in human endometrium.
Panel D : Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab317582 and ab315802 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582 , the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver labelling GLUT2 with ab260003 at 1/1000 (B), eNOS with ab317582 at 1/500 dilution (C) and CD163 with ab213612 at 1/8000 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and Nuclear DNA was labeled with DAPI (shown in blue). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-GLUT2 (magenta; Opal™690), anti-eNOS (green; Opal™520) and anti-CD163 (gray; Opal™570) on human liver.
Panel B : anti-GLUT2 staining membrane of hepatocytes in human liver.
Panel C : anti-eNOS staining endothelium in human liver.
Panel D : anti-CD163 staining Kupffer cells in human liver.

The section was incubated in three rounds of staining : in the order of ab260003, ab317582 and ab213612 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human liver tissue staining eNOS with ab317582 at a 1 : 500 (1.032 ug/ml) dilution, ab315802 anti-CD34 used at 1 : 2000 (0.26 ug/ml) dilution.

Panel A : merged staining of anti-eNOS (magenta; Opal™520), anti-CD34 (green; Opal™570) on human liver.
Panel B : anti-eNOS staining endothelium in human liver.
Panel C : anti-CD34 staining endothelium in human liver.
Panel D : Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab317582 and ab315802 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human liver tissue staining eNOS with ab317582 at a 1 : 1000 (1.004 ug/ml) dilution, ab317582 anti-eNOS used at 1 : 500 (1.032 ug/ml) dilution and ab213612 anti-CD163 used at a 1 : 8000 (0.13 ug/ml) dilution.

Panel A : merged staining of anti-GLUT2 (magenta; Opal™690), anti-eNOS (green; Opal™520) and anti-CD163 (gray; Opal™570) on human liver.
Panel B : anti-GLUT2 staining membrane of hepatocytes in human liver.
Panel C : anti-eNOS staining endothelium in human liver.
Panel D : anti-CD163 staining Kupffer cells in human liver.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab260003, ab317582 and ab213612 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HUVEC (human umbilical vein endothelial cell) cells labelling eNOS with ab317582 at 1/100 (5.16 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing cytoplasmic and membranous staining in HUVEC cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control : HeLa (PMID : 19559671)
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). 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 2 ug/ml dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa(human cervical adenocarcinoma epithelial cell, Left) / HUVEC(human umbilical vein endothelial cell, Right) cells labelling eNOS with ab317582 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : HeLa

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human tonsil tissue staining eNOS with ab317582 at a 1 : 500 (1.032 ug/ml) dilution, ab315802 anti-CD34 used at 1 : 2000 (0.26 ug/ml) dilution.

Panel A : merged staining of anti-eNOS (magenta; Opal™520), anti-CD34 (green; Opal™570) on human tonsil.
Panel B : anti-eNOS staining endothelium in human tonsil.
Panel C : anti-CD34 staining endothelium in human tonsil.
Panel D : Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab317582 and ab315802 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7(mouse Abelson murine leukemia virus-induced tumor macrophage, Left) / bEnd.3(mouse brain endothelial cell, Right) cells labelling eNOS with ab317582 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : RAW 264.7

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat skeletal muscle tissue labeling eNOS with ab317582 at 1/200 (2.58 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : no staining on rat skeletal muscle. The section was incubated with ab317582 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-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling eNOS with ab317582 at 1/200 (2.58 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on endothelial cells in rat spleen. The section was incubated with ab317582 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-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized bEnd.3 (mouse brain endothelial cell) cells labelling eNOS with ab317582 at 1/100 (5.16 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing cytoplasmic and membranous staining in bEnd.3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control : RAW 264.7 (PMID : 18607531)
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). 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 2 ug/ml dilution.

• WB

Supplier Data

Western blot - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Low expression : skeletal muscle.

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

All lanes:

Western blot - Anti-eNOS antibody [RM1181] (<a href='/en-us/products/primary-antibodies/enos-antibody-rm1181-ab317582'>ab317582</a>) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

Human placenta tissue lysate at 20 µg

Lane 3:

Human skeletal muscle 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: 35 kDa,60 kDa,100 kDa,140 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-eNOS antibody [RM1181] - BSA and Azide free (AB317583)

This data was developed using ab317582, the same antibody clone in a different buffer formulation.

Negative controls : HeLa (PMID : 19559671) and RAW 264.7 (PMID : 18607531).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 16195740 and PMID : 11331296).

This antibody was tested on fresh and frozen HUVEC and bEnd.3 whole Cell Lysate.

The Lysates in lanes 6-7 were freshly made and used for Western Blotting immediately to minimize protein degradation.

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

Exposure time : Lane 1-3 : 15 seconds Lane 4-5 : 59 seconds Lane 6 : 26 seconds Lane 7 : 180 seconds

All lanes:

Western blot - Anti-eNOS antibody [RM1181] (<a href='/en-us/products/primary-antibodies/enos-antibody-rm1181-ab317582'>ab317582</a>) at 1/1000 dilution

Lanes 1 and 6:

HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lane 2:

EA.hy926 (human somatic cell hybrid endothelial cell) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 4 and 7:

bEnd.3 (mouse brain endothelial cell) whole cell lysate at 20 µg

Lane 5:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) 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: 35 kDa,60 kDa,100 kDa,140 kDa,36 kDa

false