Anti-eNOS antibody [EPR23750-3]

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-eNOS antibody [EPR23750-3] (AB252439)

Fluorescence multiplex immunohistochemical analysis of human liver (formalin-fixed paraffin-embedded section). Panel A shows merged staining of anti-MASP2 stained on cytoplasm of hepatocytes (ab277520; gray; Opal™690) at 1 : 100 ( 5.22 μg/ml) [Panel B] , anti-CD163 stained on Kupffer cells (ab213612; green; Opal™520) at 1 : 8000 ( 0.13 μg/ml) [Panel C], and anti-eNOS tained on endothelial cells (ab252439; red; Opal™570) at 1 : 200 ( 3.005 μg/ml) [Panel D] on human liver. DAPI was used as a nuclear counter stain. Followed by Opal Polymer HRP Ms + Rb secondary. 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. The section was incubated in three rounds of staining : in the order of ab277520, ab213612, and ab252439 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-eNOS antibody [EPR23750-3] (AB252439)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human cervix adenocarcinoma epithelial cell, left) /HUVEC (human umbilical vein endothelial cell, right) cells labelling eNOS with ab252439 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : Hela (PMID : 19559671).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (AB252439)

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling eNOS with ab252439 at 1/500 (1.198 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in human placenta. The section was incubated with ab252439 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-eNOS antibody [EPR23750-3] (AB252439)

Fluorescence multiplex immunohistochemical analysis of human liver (formalin-fixed paraffin-embedded section). Panel A shows merged staining of anti-eNOS stained on endothelial cells (ab252439; red; Opal™570) at 1 : 1000 ( 1.004 μg/ml) [Panel B], anti-CD163 stained on Kupffer cells (ab213612; green; Opal™520) at 1 : 8000 ( 0.13 μg/ml) [Panel B], and anti-Glucose Transporter GLUT2 stained on membrane of hepatocytes (ab234440; gray; Opal™690) at 1 : 200 ( 3.005 μg/ml) [Panel D] on human liver. DAPI was used as a nuclear counter stain. Followed by Opal Polymer HRP Ms + Rb secondary. 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. The section was incubated in three rounds of staining : in the order of ab234440, ab213612, and ab252439 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (AB252439)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue labeling eNOS with ab252439 at 1/500 (1.198 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on endothelial cells in human lung carcinoma. The section was incubated with ab252439 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (AB252439)

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling eNOS with ab252439 at 1/500 (1.198 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on endothelial cells in human spleen. The section was incubated with ab252439 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (AB252439)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling eNOS with ab252439 at 1/500 (1.198 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on endothelial cells in human kidney. The section was incubated with ab252439 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-eNOS antibody [EPR23750-3] (AB252439)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HUVEC cells labelling eNOS with ab252439 at 1/100 (5.99 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HUVEC cells. Negative control : HeLa (PMID : 19559671). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• IP

Unknown

Immunoprecipitation - Anti-eNOS antibody [EPR23750-3] (AB252439)

eNOS was immunoprecipitated from 0.35 mg EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate with ab252439 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab252439 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate 10 ug

Lane 2 : ab252439 IP in EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab252439 in EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate

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

Exposure time : 6 seconds.

IP lysates were unboiled.

All lanes:

Immunoprecipitation - Anti-eNOS antibody [EPR23750-3] (ab252439)

Predicted band size: 133 kDa

Observed band size: 140 kDa

false

• WB

Lab

Western blot - Anti-eNOS antibody [EPR23750-3] (AB252439)

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

HUVEC whole cell lysate in lane 1 is made freshly and used in WB test immediately to minimize protein degradation.

Exposure time : 6 seconds.

All lanes:

Western blot - Anti-eNOS antibody [EPR23750-3] (ab252439) at 1/1000 dilution

All lanes:

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

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 133 kDa

Observed band size: 140 kDa

false

• WB

Lab

Western blot - Anti-eNOS antibody [EPR23750-3] (AB252439)

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

Negative control : HeLa (PMID : 19559671).

Lysates were made freshly and used in WB test immediately to minimize protein degradation. Lysates were unboiled.

Exposure time : 6 seconds.

All lanes:

Western blot - Anti-eNOS antibody [EPR23750-3] (ab252439) at 1/1000 dilution

Lane 1:

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

Lane 2:

HeLa (human cervix 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

Predicted band size: 133 kDa

Observed band size: 140 kDa

false