All lanes : Anti-IL-33 antibody [EPR17831] (ab187060) at 1/1000 dilution

Lane 1 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : RAW 264.7 treated with 50 nM Phorbol-12-myristate-13-acetate (PMA) and 5 ug/ml lipopolysaccharide (LPS) for 24 hours, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Developed using the ECL technique.

Predicted band size: 30 kDa
Observed band size: 33 kDa why is the actual band size different from the predicted?

Blocking/Dilution: 5% NFDM/TBST

Exposure: 3 minutes

IL-33 expression is induced by LPS treatment of PMA-differentiated RAW 264.7 cells (PMID 19559631; PMID 19933859).

All lanes : Anti-IL-33 antibody [EPR17831] (ab187060) at 1/1000 dilution

Lane 1 : Rat lung tissue lysate at 20 µg
Lane 2 : Mouse lung tissue lysate at 10 µg

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Developed using the ECL technique.

Predicted band size: 30 kDa
Observed band size: 30 kDa

Blocking/Dilution: 5% NFDM/TBST

Exposure: 3 minutes

Flow Cytometry analysis of RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 50nM PMA and 5μg/ml LPS for 24h (Red) / Untreated control (Green) labeling IL-33 with ab187060 at 1/500 dilution. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody. Cells were fixed with 4% paraformaldehyde and permeabilised with 0.1% Tween-20. Isotype control - Rabbit monoclonal IgG (ab172730) (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling IL33 with ab187060 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), ready to use. Nuclear staining in endothelial cells of mouse spleen is observed (PMID: 12819012). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), ready to use.

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

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling IL33 with ab187060 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), ready to use. Nuclear staining in endothelial cells of rat spleen is observed (PMID: 12819012). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), ready to use.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling IL33 with ab187060 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased nuclear staining in RAW 264.7 cells treated with 50 nM Phorbol-12-myristate-13-acetate (PMA) and 5 µg/ml Lipopolysaccharide for 24h.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.