重组Anti-eNOS抗体[RM1181] - BSA and Azide free (ab317583)

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

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

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

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

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

Low expression: skeletal muscle.

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

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

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

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

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

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

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

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 (reen; 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.

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 (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.

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 stomach 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 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.

Nuclear counter stain with DAPI.

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

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.

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 endometrium 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 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.

Nuclear counter stain with DAPI.

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

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.

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.

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

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