Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized fresh mouse spleen tissue labeling IL-6 with ab290735 at 1/50 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary at 1/1000. Increased staining on mouse spleen treated with lipopolysaccharide (1 μg/mL) and Brefeldin A (1 μg/mL) for 16 h. The nuclear counterstain was DAPI (Blue).

Immunohistochemical analysis of paraffin-embedded Mouse spleen treated tissue labelling IL-6 with ab290735 at 1/100 (5.75 ug/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection)

Positive staining on mouse spleen treated with lipopolysaccharides (1 ug/ml) for 16 h in vitro (A) and no staining on untreated mouse spleen (B). The section was incubated with ab290735 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).

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

Immunohistochemical analysis of paraffin-embedded Mouse lung treated tissue labelling IL-6 with ab290735 at 1/100 (5.75 ug/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on Mouse lung treated with lipopolysaccharides (1 ug/ml) for 16 h in vitro (A) and no staining on untreated mouse lung (B). The section was incubated with ab290735 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).

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

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T cells transfected with mouse IL-6 expression vector containing a his tag, labelling IL-6 with ab290735 at 1/100 (5.75 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on HEK-293T cells transfected with mouse IL-6 expression vector containing a his tag (A). No staining on HEK-293T cells transfected with empty vector containing a his tag (B). The section was incubated with ab290735 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).

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

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

Lanes 1-2 : Anti-IL-6 antibody [EPR23819-103] (ab290735) at 1/1000 dilution
Lanes 3-4 : Anti-IL-6 antibody [EPR21710] (ab229381) at 1/1000 dilution
Lanes 5-6 : Anti-IL-6 antibody [EPR23819-11] (ab259341) at 1/1000 dilution

The expression profile/molecular weight observed is consistent with that described in the literature (PMID:2523818, PMID:25130514).
IL-6 expressed in LPS-induced RAW264.7 cells (PMID:31718640, PMID:30823686).

Compared with ab290735, ab229381 and ab259341 have higher affinity. We recommend ab229381 or ab259341 as alternatives for western blot.

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

The expression profile/molecular weight observed is consistent with that described in the literature (PMID:2523818, PMID:25130514).
IL6 expressed in LPS-induced RAW264.7 cells (PMID:31718640, PMID:30823686).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells lebelling IL-6 with ab290735 at 1/50 (11.5 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2ug/ml dilution) (Green). Confocal image showing cytoplasmic staining in RAW 264.7 cells treated with LPS (100 ng/ml) for 4h, with addition of BFA (1 ug/ml) for the last 3 hours is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5ug/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 (2ug/ml) dilution.