Recombinant Anti-IL-1 alpha antibody [EPR25263-27] (ab300499)

Blocking / Diluting buffer and concentration: 5% NFDM/TBST

The expression level of IL-1 alpha was upregulated by LPS stimulation (PMID: 25870118)

Immunohistochemical analysis of paraffin-embedded Mouse lung treated tissue labeling IL-1 alpha with ab300499 at 1/500 dilution (1.05 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on mouse lung induced by LPS+BFA (A) and no staining on untreated mouse lung (B). The section was incubated with ab300499 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labeling IL-1 alpha with ab300499 at 1/50 (10.5 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 2 µg/mL dilution (Green). Confocal image showing increased nuclear staining in THP-1 cells treated with Phorbol-12-myristate-13-acetate (50 nM) for 24 h. is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 2µg/mL dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) cells labeling IL-1 alpha with ab300499 at 1/50 (10.5 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 2µg/mL dilution (Green). Confocal image showing increased nuclear staining in Raw 264.7 cells treated with lipopolysaccharide (10 µg/ml) for 24 h (PMID:25870118) is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). 

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 2µg/mL dilution.

Flow Cytometry (Intracellular) analysis of mouse bone marrow cells treated with 5 µg/ml LPS for 7 hours (Right) / Untreated control (Left) labelling IL-1 alpha with ab300499 at a 1/500 dilution. Cells were fixed with 2% paraformaldehyde and permeabilized using True-Nuclear buffer. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution. Cells were surface stained with BV421 conjugated CD11b, then fixed with PFA followed by intracellularly stained with ab300499.

Flow Cytometry (Intracellular) analysis of RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 10 µg/ml LPS for 24 hours (Right) / Untreated control (Left) IL-1 alpha with ab300499 at a 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat Anti-Rabbit IgG (Alexa Fluor® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution. The expression level of IL-1 alpha was upregulated by LPS stimulation (PMID: 25870118).