Anti-CXCL10/IP-10 antibody [EPR24674-84]

5 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCL10/IP-10 antibody [EPR24674-84] (ab306587)

  Immunohistochemical analysis of paraffin-embedded (A) THP-1 (human mon tissue labeling CXCL10/IP-10 with ab306587 at 1/2000 (0.248 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on (A) THP-1 (human monocytic leukemia monocyte) treated with 200ng/ml IFN-y (Recombinant Human Interferon gamma protein (Active) ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5µg/ml
  
  Brefeldin A for the last 21 hours cell pellet. No staining on (B) Untreated THP-1 cell pellet. The section was incubated with ab306587 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution.
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCL10/IP-10 antibody [EPR24674-84] (ab306587)

  Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling CXCL10/IP-10 with ab306587 at 1/2000 (0.248 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on human colonic immune cells (PMID: 10433925; PMID: 24748971).The section was incubated with ab306587 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution.
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCL10/IP-10 antibody [EPR24674-84] (ab306587)

  Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CXCL10/IP-10 with ab306587 at 1/2000 (0.248 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on human tonsil (PMID: 12949239).The section was incubated with ab306587 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution.
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

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  Immunoprecipitation - Anti-CXCL10/IP-10 antibody [EPR24674-84] (ab306587)

  CXCL10/IP-10 was immunoprecipitated from 0.35 mg THP-1 treated with 200ng/ml IFN-y (Recombinant Human Interferon gamma protein (Active) ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5μg/ml Brefeldin A for the last 21 hours whole cell lysate 20 μg with ab306587 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab306587. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  
  Lane 1: THP-1 treated with 200ng/ml IFN-y (Recombinant Human Interferon gamma protein (Active) ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5μg/ml Brefeldin A for the last 21 hours whole cell lysate 20 μg
  
  Lane 2: ab306587 IP in THP-1 treated with 200ng/ml IFN-y (Recombinant Human Interferon gamma protein (Active) ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5μg/ml Brefeldin A for the last 21 hours
  
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab306587 in THP-1 treated with 200ng/ml IFN-y (Recombinant Human Interferon gamma protein (Active) ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5μg/ml Brefeldin A for the last 21 hours whole cell lysate 20 μg
  
  
  Blocking and dilution buffer and concentration: 5% NFDM/TBST.
  
  Exposure time: 180 seconds

  All lanes: Immunoprecipitation - Anti-CXCL10/IP-10 antibody [EPR24674-84] (ab306587) at 1/30 dilution

  All lanes: THP-1

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 11 kDa

  Exposure time: 180s

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  Western blot - Anti-CXCL10/IP-10 antibody [EPR24674-84] (ab306587)

  Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
  
  False colour image of Western blot: Anti-CXCL10/IP-10 antibody [EPR24674-84] (ab306587) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red.
  In Western blot, ab306587 was shown to bind specifically to CXCL10/IP-10. A band was observed at 11 kDa in wild-type A549 cell lysates with no signal observed at this size in CXCL10/IP-10 knockout cell line Human CXCL10 (IP10) knockout A549 cell line ab266971 (knockout cell lysate Human CXCL10 (IP10) knockout A549 cell lysate ab256888). To generate this image, wild-type and CXCL10/IP-10 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/10000 dilution.

  All lanes: Western blot - Anti-CXCL10/IP-10 antibody [EPR24674-84] (ab306587) at 1/1000 dilution

  Lane 1: Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

  Lane 2: THP-1 treated with /ml IFN-y (Recombinant Human Interferon gamma protein (Active) ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5ug/ml Brefeldin A for the last 21 hours whole cell lysate at 20 µg

  Lane 3: Untreated Wild-type A549 (human lu carcinoma epithelial cell) whole cell lysate 40 at 40 µg

  Lane 4: Wild-type A549 treated with /ml IFN-y (Recombinant Human Interferon gamma protein (Active) ab259377) for 32 hours and 10 ng/m TNF-alpha (Recombinant human TNF alpha protein (Active) ab259410) for 32 hours, and 5ug/ml Brefeldin A (Brefeldin A, Inhibitor of ADP-ribosylation factor ab120299) for the last 6 hours whole cell lysate 40 at 40 µg

  Lane 5: Untreated CXCL10/IP-10 knockout A549 whole cell lysate at 40 µg

  Lane 6: CXCL10/IP-10 knockout A549 treated with /ml IFN-y (Recombinant Human Interferon gamma protein (Active) ab259377) for 32 hours and 10 ng/m TNF-alpha (Recombinant human TNF alpha protein (Active) ab259410) for 32 hours, and 5ug/ml Brefeldin A (Brefeldin A, Inhibitor of ADP-ribosylation factor ab120299) for the last 6 hours whole cell lys at 40 µg

  Secondary

  Lanes 1 - 6: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution

  Lanes 1 - 6: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/10000 dilution

  Observed band size: 11 kDa