Anti-IL-6 antibody [EPR23819-103]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-6 antibody [EPR23819-103] (AB290735)

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

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-IL-6 antibody [EPR23819-103] (AB290735)

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).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-6 antibody [EPR23819-103] (AB290735)

Immunohistochemical analysis of formalin fixed paraffin embedded lipopolysaccharide (1 ug/ml) treated mouse lung labelling IL-6 with ab290735 at a concentration of 4µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a ChromoMap DAB kit and anti-rabbit HQ and anti-HQ HRP detection. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab290735 Anti-IL-6 antibody [EPR23819-103] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in untreated tissues. Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IL-6 antibody [EPR23819-103] (AB290735)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-6 antibody [EPR23819-103] (AB290735)

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-6 antibody [EPR23819-103] (AB290735)

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

• WB

Supplier Data

Western blot - Anti-IL-6 antibody [EPR23819-103] (AB290735)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. The expression of IL-10 is upregulated in response to LPS in treated RAW 264.7 (PMID : 23012430, 15218058) but not in non-differentiated NIH/3T3 cells. In western blot, anti-IL-6 antibody (ab290735) staining at 1/1000 dilution. Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-IL-10 antibody [EPR27280-88] (<a href='/en-us/products/primary-antibodies/il-10-antibody-epr27280-88-ab310329'>ab310329</a>) at 1/1000 dilution

Lane 1:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW264.7 treated with 100 ng/mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate at 20 µg

Lane 3:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 treated with 100 ng/mL LPS for 6 hours, 300ng/mL Brefeldin A was then added for additional 3 hours whole cell lysate 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: 20 kDa

Observed band size: 18 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-IL-6 antibody [EPR23819-103] (AB290735)

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.

All lanes:

Western blot - Anti-IL-6 antibody [EPR23819-103] (ab290735) at 1/1000 dilution

Lanes 1, 3 and 5:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lanes 2, 4 and 6:

RAW264.7 treated with 0.1µg/ml lipopolysaccharide (LPS) for 4 hours, then with 1 µg/ml Brefeldin A (BFA) added for 3 hours, whole cell lysate 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: 23 kDa

Observed band size: 24 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-IL-6 antibody [EPR23819-103] (AB290735)

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).

All lanes:

Western blot - Anti-IL-6 antibody [EPR23819-103] (ab290735) at 1/1000 dilution

Lane 1:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate. at 20 µg

Lane 2:

RAW264.7 treated with 0.1µg/ml lipopolysaccharide (LPS) for 4 hours, then with 1 µg/ml Brefeldin A (BFA) added for 3 hours whole cell lysate. 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 1/20 dilution

Predicted band size: 23 kDa

Observed band size: 24 kDa

false

Exposure time: 3min