Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling Heme Oxygenase 1 with ab52947 at 1/50 dilution (1ug) (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). A Goat anti rabbit IgG (Alexa Fluor 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling Heme Oxygenase 1 with ab52947 at 1/2000 (0.246 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. Cytoplasmic staining on human spleen. The section was incubated with ab52947 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) was used.

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

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Heme Oxygenase 1 with ab52947 at 1/2000 (0.246 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. Cytoplasmic staining on mouse spleen. The section was incubated with ab52947 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) was used.

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

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Heme Oxygenase 1 with ab52947 at 1/2000 (0.246 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. Cytoplasmic staining on Kupffer cells in human liver. The section was incubated with ab52947 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) was used.

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

All lanes : Anti-Heme Oxygenase 1 antibody [EP1391Y] (ab52947) at 1/1000 dilution

Lane 1 : Hek293
Lane 2 : HL60
Lane 3 : HeLa
Lane 4 : A549
Lane 5 : Hu spleen
Lane 6 : Ms spleen
Lane 7 : Rt spleen

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye® 800CW Goat anti Rabbit

Predicted band size: 33 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

Hek293 & HL60 presumed negative or very low expression.

Loading control GAPDH at 38kDa

IHC image of ab52947 staining in mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab52947, 5µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Heme Oxygenase 1 was immunoprecipitated from 0.35mg mouse spleen lysate with ab52947 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab52947 at 1/1000 dilution (1 μg/mL). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/1000 dilution.

Lane 1: Mouse spleen tissue lysate 10 μg
Lane 2: Mouse spleen tissue lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab52947 in mouse spleen lysate.

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

Overlay histogram showing HEK-293 (human epithelial cell line from embryonic kidney) cells stained with ab52947 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52947, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HEK293 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-Heme Oxygenase 1 antibody [EP1391Y] (ab52947) at 1/1000 dilution

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysates with 5% NFDM/TBST
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates with 5% NFDM/TBST
Lane 3 : A549 (Human lung carcinoma epithelial cell) whole cell lysates with 5% NFDM/TBST
Lane 4 : Mouse spleen lysates with 5% NFDM/TBST

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 33 kDa
Observed band size: 33 kDa


Exposure time: 120 seconds

We are unsure how to define the extra bands