Anti-TNF alpha antibody [EPR21753-109]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [EPR21753-109] (AB205587)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse cerebrum labelling TNF alpha with ab205587 at a concentration of 1/1000 dilution. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The primary antibody Anti-TNF alpha antibody [EPR21753-109] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Negative control : no staining on mouse cerebrum.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [EPR21753-109] (AB205587)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse lung labelling TNF alpha with ab205587 at a concentration of 1/1000 dilution. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The primary antibody Anti-TNF alpha antibody [EPR21753-109] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Positive staining on mouse lung treated with LPS (1ug/ml for 16 hours) and BFA (1ug/ml for 16h hours) (Image A) and no staining on wild type mouse lung (Image B).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-TNF alpha antibody [EPR21753-109] (AB205587)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) (+/- treatment with 100ng/ml LPS for 3h, then together with 300ng/ml BFA for another 3h) cells labeling TNF alpha with ab205587 at 1/100 dilution, followed by a AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in RAW 264.7 cells treated with 100ng/ml LPS for 3h, then together with 300ng/ml BFA for another 3h. The Nuclear counterstain is DAPI (Blue). Tubulin was stained using an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594, ab195889) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-TNF alpha antibody [EPR21753-109] (AB205587)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed 90% methanol-permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 3h, then together with 300ng/ml BFA for another 3h (Red) / Untreated control (Green), compared to an unlabeled control (cells without incubation with primary antibody and secondary antibody, Blue) and an isotype control- Rabbit monoclonal IgG (ab172730, Black). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077)at 1/2000 dilution was used as the secondary antibody.

• IP

Unknown

Immunoprecipitation - Anti-TNF alpha antibody [EPR21753-109] (AB205587)

TNF alpha was immunoprecipitated from 0.35 mg rat splenocytes (treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM BFA for 6h) whole cell lysate with ab205587 at 1/50 dilution. Western blot was performed on the immunoprecipitate using ab205587 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1 : Rat splenocytes (treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM BFA for 6h) whole cell lysate 10μg (input).

Lane 2 : ab205587 IP in rat splenocytes (treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM BFA for 6h) whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab205587 in rat splenocytes (treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM BFA for 6h) whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 30 seconds.

The MW observed is consistent with what has been described in the literature (PMID : 9933416).

All lanes:

Immunoprecipitation - Anti-TNF alpha antibody [EPR21753-109] (ab205587)

Predicted band size: 25 kDa

false

• WB

Lab

Western blot - Anti-TNF alpha antibody [EPR21753-109] (AB205587)

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

IL-1 beta and IL-6 could be a good control validating the increased TNF alpha level after drug treatment.

Lanes 1 - 6:

Western blot - Anti-TNF alpha antibody [EPR21753-109] (ab205587) at 1/1000 dilution

Lanes 1 - 6:

Western blot - Anti-TNF alpha antibody [EPR21753-109] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/tnf-alpha-antibody-epr21753-109-bsa-and-azide-free-ab252382'>ab252382</a>) at 1/1000 dilution

Lanes 1, 3 and 5:

Untreated NR8383 (rat lung macrophage (alveolar)) whole cell lysate at 20 µg

Lane 2:

NR8383 (rat lung macrophage (alveolar)) treated with 100ng/ml lipopolysaccharide (LPS) for 4h then add 1000ng/ml BFA for another 3h whole cell lysate at 20 µg

Lane 4:

NR8383 (rat lung macrophage (alveolar)) treated with 100ng/ml lipopolysaccharide (LPS) for 3h then add 300ng/ml BFA for another 3h whole cell lysate at 20 µg

Lane 6:

NR8383 (rat lung macrophage (alveolar)) treated with 1000ng/ml lipopolysaccharide (LPS) for 2h 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

Observed band size: 23 kDa,26 kDa

false

Exposure time: 20s

• WB

Unknown

Western blot - Anti-TNF alpha antibody [EPR21753-109] (AB205587)

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

Exposure time : 20 seconds

The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID : 9933416)

All lanes:

Western blot - Anti-TNF alpha antibody [EPR21753-109] (ab205587) at 1/1000 dilution

Lane 1:

Untreated rat splenocytes whole cell lysate at 20 µg

Lane 2:

Rat splenocytes treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM BFA for 6h whole cell lysate at 20 µg

Lane 3:

Untreated RAW 264.7(mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 4:

RAW 264.7 treated with 100ng/ml LPS for 3h, then together with 300ng/ml BFA for another 3h 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 1/100000 dilution

Predicted band size: 25 kDa

false

• WB

Lab

Western blot - Anti-TNF alpha antibody [EPR21753-109] (AB205587)

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

Normal tissues express undetectable level of TNF alpha, IL-1 beta and IL-6 proteins.

IL-1 beta and IL-6 could be a good control validating the increased TNF alpha level after drug treatment.

All lanes:

Western blot - Anti-TNF alpha antibody [EPR21753-109] (ab205587) at 1/1000 dilution

Lane 1:

Rat cerebral cortex tissue lysate at 20 µg

Lane 2:

Rat cerebellum tissue lysate at 20 µg

Lane 3:

Rat hippocampus tissue lysate at 20 µg

Lane 4:

Rat spinal cord tissue lysate at 20 µg

Lane 5:

Rat small intestine tissue lysate at 20 µg

Lane 6:

Rat kidney tissue lysate at 20 µg

Lane 7:

Rat skin tissue lysate at 20 µg

Lane 8:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 9:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml lipopolysaccharide (LPS) for 4h then add 1000ng/ml BFA for another 3h 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

Observed band size: 23 kDa,26 Da

false

Exposure time: 180s

• WB

CiteAb

Western blot - Anti-TNF alpha antibody [EPR21753-109] (AB205587)

TNF alpha western blot using anti-TNF alpha antibody [EPR21753-109] ab205587. Publication image and figure legend from Tang, D. S., Cao, F., et al., 2022, Front Immunol, PubMed 35911777.

ab205587 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab205587 please see the product overview.

Macrophages overexpressing miR-183-5p aggravate AC damage. (A) Representative western blot and quantification of P-P65/P65 and TNF-α levels in ACs from the four groups : Ctrl, Cae, Cae+NC-mimic M, and Cae+miR-183-5p mimic M. (B) Trypsinogen activation assay in ACs from the above groups. The percentage of positive cells (white arrow) was calculated to quantify the degree of trypsinogen activation. (C) Representative flow cytometry results for necrosis in ACs from the above groups. All experiments were repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001. Ctrl : PBS-treated acinar cells, Cae : caerulein-treated acinar cells, Cae+NC-mimic : M caerulein-treated acinar cells cocultured with macrophages transfected with the NC-mimic, Cae+miR-183-5p mimic M : caerulein-treated acinar cells cocultured with macrophages transfected with the miR-183-5p mimic.

false