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.

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.

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).

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.

Anti-eNOS antibody [EPR23750-3] (ab252439) at 1/1000 dilution + HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 133 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

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.

 

Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue labeling eNOS with abab252439 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.

All lanes : Anti-eNOS antibody [EPR23750-3] (ab252439) at 1/1000 dilution

Lane 1 : EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 133 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

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.

 

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.

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.