Anti-Heme Oxygenase 1 antibody [EP1391Y]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

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

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

Intracellular 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

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

• IP

Lab

Immunoprecipitation - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

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.

All lanes:

Immunoprecipitation - Anti-Heme Oxygenase 1 antibody [EP1391Y] (ab52947)

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

Lanes 1 - 7 : Merged signal (red and green). Green - ab52947 observed at 33 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab52947 was shown to react with Heme Oxygenase 1 in wild-type A549 cells in Western blot with loss of signal observed in HMOX1 knockout cell line ab269503 (knockout cell lysate ab269665). Wild-type A549 and HMOX1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab52947 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Heme Oxygenase 1 antibody [EP1391Y] (ab52947) at 1/2000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

HMOX1 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human HMOX1 (Heme Oxygenase 1) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-hmox1-heme-oxygenase-1-knockout-a549-cell-line-ab269503'>ab269503</a>)

Lane 3:

Human Spleen tissue lysate at 20 µg

Lane 4:

HL-60 cell lysate at 20 µg

Lane 5:

MCF7 cell lysate at 20 µg

Lane 6:

HeLa cell lysate at 20 µg

Lane 7:

A549 cell lysate at 20 µg

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

• WB

Lab

Western blot - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

Hek293 & HL60 presumed negative or very low expression.

Loading control GAPDH at 38kDa

All lanes:

Western blot - Anti-Heme Oxygenase 1 antibody [EP1391Y] (ab52947) at 1/1000 dilution

Lane 1:

Hek293 at 10 µg

Lane 2:

HL60 at 10 µg

Lane 3:

HeLa at 10 µg

Lane 4:

A549 at 10 µg

Lane 5:

Hu spleen at 10 µg

Lane 6:

Ms spleen at 10 µg

Lane 7:

Rt spleen at 10 µg

Secondary

All lanes:

IRDye® 800CW Goat anti Rabbit

Predicted band size: 33 kDa

Observed band size: 32 kDa

false

• WB

Lab

Western blot - Anti-Heme Oxygenase 1 antibody [EP1391Y] (AB52947)

We are unsure how to define the extra bands.

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

All lanes:

Western blot - 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 at 20 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Lane 3:

A549 (Human lung carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 4:

Mouse spleen lysates 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 1/20000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

Exposure time: 120s