重组Anti-CXCL10/IP-10抗体[RM2054] (ab318282)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

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

In Western blot, ab318282 was shown to bind specifically to CXCL10/IP-10.  Target of interest was observed at 11 kDa in wild-type A549 cell lysates treated with 100 ng/ml IFN gamma (ab259377) for 32 hours and 10 ng/m TNF alpha (ab259410) for 32 hours, and 5 ug/ml Brefeldin A (ab120299) for the last 6 hours (lane 2) with no signal observed at this size in CXCL10/IP-10 knockout cell line (lane 3-4) (lane 3, knockout cell line ab266971 / knockout cell lysate ab256888).

The identity of the bands between 35 kDa and 75 kDa are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

Immunohistochemical analysis of paraffin-embedded Human appendix tissue labeling CXCL10/IP-10 with ab318282 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Positive staining on human appendix immune cells (PMID: 10433925; PMID: 24748971).
The section was incubated with ab318282 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 was performed with citrate buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CXCL10/IP-10 with ab318282 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Positive staining on human tonsil (PMID: 12949239).
The section was incubated with ab318282 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 was performed with citrate buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded (A) THP-1 (human monocytic leukemia monocyte) treated with 200ng/ml IFN-y (ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5ug/ml Brefeldin A for the last 21 hours cell pellet (B) Untreated THP-1 cell pellet tissue labeling CXCL10/IP-10 with ab318282 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Positive staining on (A) THP-1 (human monocytic leukemia monocyte) treated with 200ng/ml IFN-y for 24 hours and 50ng/ml LPS for 24 hours, and 5ug/ml Brefeldin A for the last 21 hours cell pellet. No staining on (B) Untreated THP-1 cell pellet.
The section was incubated with ab318282 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 was performed with citrate buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling CXCL10/IP-10 with ab318282 at 1/2000 (0.25 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Negative control: No staining on human cerebrum (PMID: 14507644).
The section was incubated with ab318282 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 was performed with citrate buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CXCL10 KO A549 (human lung carcinoma epithelial cell) cells labelling CXCL10/IP-10 with ab318282 at 1/50 (9.98 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green).

Confocal image showing cytoplasmic staining in parental A549 cells treated with INF-γ (100 ng/mL) and TNF-α (10 ng/mL) for 32 h, with addition of brefeldin A (5 ug/mL) for the last 6 h (shown in green), and negative staining in CXCL10 knockout A549 cells with the same condition. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
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 2.5ug/ml dilution (Magenta). 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 ug/mL dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labelling CXCL10/IP-10 with ab318282 at 1/50 (9.98 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green).

Confocal image showing cytoplasmic staining in THP-1 cells treated with INF-γ (200 ng/mL) and Lipopolysaccharides (50 ng/mL) for 24 h add Brefeldin A (1ug/ml) for 21 h (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
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 2.5ug/ml dilution (Magenta). 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 ug/mL dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1 (human monocytic leukemia monocyte) treated with 200ng/ml 3+21h and 50ng/ml 3+21h IFN-r, LPS then add 1ug/ml 21h BFA (green) /Untreated with THP-1 (magenta) cells labelling CXCL10/IP-10 with ab318282 at 1/50 dilution (1ug) / Green and magenta compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor ® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

CXCL10/IP-10 was immunoprecipitated from 0.35 mg THP-1(human monocytic leukemia monocyte) treated with 200 ng/ml INF gamma (ab259377) and 50 ng/ml LPS for 24 hours, and 5 ug/ml Brefeldin A (ab120299) for the last 21 hours whole cell lysate with ab318282 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab318282 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1(human monocytic leukemia monocyte) treated with 200 ng/ml INF gamma (ab259377) and 50 ng/ml LPS for 24 hours, and 5 ug/ml Brefeldin A (ab120299) for the last 21 hours whole cell lysate
Lane 2: ab318282 IP in THP-1(human monocytic leukemia monocyte) treated with 200 ng/ml INF gamma (ab259377) and 50 ng/ml LPS for 24 hours, and 5 ug/ml Brefeldin A (ab120299) for the last 21 hours whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab318282 in THP-1 treated with 200 ng/ml INF gamma (ab259377) and 50 ng/ml LPS for 24 hours, and 5 ug/ml Brefeldin A (ab120299) for the last 21 hours whole cell lysate

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

Exposure time: 119 seconds.