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

Soluble form (17 kDa) and multimers of soluble form (33 kDa, 51 kDa) were also observed as described in literatures (PMID: 18523283, PMID: 28426652).

Exposure time: 180 seconds

Immunohistochemical analysis of paraffin-embedded mouse lung abeling TNF alpha with ab307164 at 1/1000 (0.56 µg/ml) followed by a ready to use Leica DS9800 (Bond^® Polymer Refine Detection) kit. Positive staining on mouse lung treated with LPS (1ug/ml for 16 hours) and BFA (1ug/ml for 16h hours) (Image A) and control mouse lung (Image B). The section was incubated with ab307164 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 ready to use Leica DS9800 (Bond^® Polymer Refine Detection) kit.

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

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling TNF alpha with ab307164 at 1/1000 (0.56 µg/ml) followed by ready to use Leica DS9800 (Bond^® Polymer Refine Detection) kit. Negative control: no staining on mouse cerebrum. The section was incubated with ab307164 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 ready to use Leica DS9800 (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% Triton X-100 permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labeling TNF alpha with ab307164 at 1/500 (1.12 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 cytoplasmic staining in human PBMC treated with Phorbol-12-myristate-13-acetate (50ng/ml) and Ionomycin calcium (200ng/ml) and Brefeldin A (300ng/ml) for 4 hours. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (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 (2 µg/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) cells labeling TNF alpha with ab307164 at 1/500 (1.12 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 cytoplasmic staining in RAW 264.7 treated with lipopolysaccharide (100 ng/ml) and Brefeldin A (1 µg/ml) for 3 hours. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (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 (2 µg/ml) dilution.

Flow cytometric (Intracellular) analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized human peripheral blood mononuclear cells (PBMC) labeling TNF alpha with ab307164 at 1/500 dilution (0.1µg). Cells were treated with 50ng/ml PMA, 200ng/ml Ionomycin calcium and 300ng/ml BFA for 4h (Right) / Untreated control (Left). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Cells were surface stained with anti-CD3 conjugated to Alexa Fluor^® 647. Then fixed with 2% PFA for 10min followed by intracellularl staining with ab307164.

Flow cytometric (Intracellular) analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling TNF alpha with ab307164 at 1/500 dilution (0.1ug).  Cells were treated with 100ng/ml LPS for 3h and add 1µg/ml BFA for another 3h (Red), untreated control (Green), and compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.