ab124962 (purified) at 1/20 immunoprecipitating IL1RA in NIH/3T3 whole cell lysate.

Lane 1 (input): NIH/3T3 whole cell lysate (10µg)

Lane 2 (+): ab124962 + NIH/3T3 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab124962 in NIH/3T3 whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124962).

Clone EPR6483 (ab226101) has been successfully conjugated by Abcam. This image was generated using Anti-IL-1RA antibody [EPR6483] (Alexa Fluor® 488). Please refer to ab252007 for protocol details.

IHC image of IL-1RA staining in a section of formalin-fixed paraffin-embedded normal human kidney*.

The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer (pH9, epitope retrieval solution 2) in a BOND RX autostainer for 20 minutes. Slides were removed from the BOND RX and all following steps were performed manually. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab252007 at 1/100 dilution (shown in green). Nuclear DNA was labeled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

Clone EPR6483 (ab226101) has been successfully conjugated by Abcam. This image was generated using Anti-IL-1RA antibody [EPR6483] (Alexa Fluor® 647). Please refer to ab252008 for protocol details.

IHC image of IL-1RA staining in a section of formalin-fixed paraffin-embedded normal human kidney*.

The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer (pH9, epitope retrieval solution 2) in a BOND RX autostainer for 20 minutes. Slides were removed from the BOND RX and all following steps were performed manually. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab252008 at 1/100 dilution (shown in red). Nuclear DNA was labeled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labelling IL1RA with purified ab124962 at a dilution of 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124962).

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling IL1RA with purified ab124962 at 1/100. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124962).

Overlay histogram showing HeLa cells fixed in 4% PFA and stained with purified ab124962 at a dilution of 1 in 20 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124962).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue labelling IL1RA with purified ab124962 at a dilution of 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124962).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling IL1RA with purified ab124962 at a dilution of 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124962).

This IHC data was generated using the same anti-IL-1RA antibody clone, EPR6483, in a different buffer formulation (cat# ab124962).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling IL-1RA with unpurified ab124962 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing A431 cells stained with unpurified ab124962 (red line). The cells were fixed with 80% methanol (5 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 (unpurified ab124962, 1/100 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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124962).

Equilibrium disassociation constant (K_D)
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This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124962).