Anti-IL-8 antibody [RM1267]

8 product images

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  Indirect ELISA - Anti-IL-8 antibody [RM1267] (ab322732)

  Indirect ELISA analysis of ab322732 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1:2500 dilution dilution.

  Antigen: Human Interleukin-8.

  Antigen concentration: 1000 ng/ml

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  Flow Cytometry (Intracellular) - Anti-IL-8 antibody [RM1267] (ab322732)

  Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized IL8 KO PC-3 (IL-8 knock out human prostate adenocarcinoma epithelial cell) treated with 2ug/mL LPS for 5hours and 5ug/mL Brefeldin A for 5 hours (Magenta) or untreated with IL8 KO PC-3(Green)(Right) / PC-3 treated with 2ug/mL LPS for 5hours and 5ug/mL Brefeldin A for 5 hours(Magenta) or untreated with PC-3(Green)(Left) cells labelling IL-8 with ab322732 at 1/500 dilution (0.1ug) / Magenta and Green compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) and Grey isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

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

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  Immunoprecipitation - Anti-IL-8 antibody [RM1267] (ab322732)

  IL-8 was immunoprecipitated from 0.35 mg PC-3 (human prostate adenocarcinoma epithelial cell) treated with 2ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, whole cell lysate 20 ug with ab322732 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322732 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.

  Lane 1: PC-3 (human prostate adenocarcinoma epithelial cell) treated with 2ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, whole cell lysate
  
  Lane 2: ab322732 IP in PC-3 (human prostate adenocarcinoma epithelial cell) treated with 2ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, whole cell lysate 20 ug
  
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab322732 in PC-3 treated with 2ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, whole cell lysate 20 ug

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

  All lanes: Immunoprecipitation - Anti-IL-8 antibody [RM1267] (ab322732) at 1/30 dilution

  All lanes: PC-3 (human prostate adenocarcinoma epithelial cell) treated with 2ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, whole cell lysate

  Secondary

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

  Exposure time: 10s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-8 antibody [RM1267] (ab322732)

  Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling IL-8 with ab322732 at 1/1000 (0.491 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  Negative control: no staining on spleen. The section was incubated with ab322732 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 Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  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-IL-8 antibody [RM1267] (ab322732)

  Immunohistochemical analysis of paraffin-embedded (A) Untreated Wild-type PC-3 (human prostate adenocarcinoma epithelial cell) cell pellet. (B) Wild-type PC-3 treated with 2ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, cell pellet (C) Untreated CXCL8 knockout PC-3 cell pellet (D) CXCL8 knockout PC-3 treated with 2ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, cell pellet tissue labeling IL-8 with ab322732 at 1/8000 (0.061 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

  Weak staining on (A) untreated Wild-type PC-3 cell pellet, positive staining on (B) wild-type PC-3 treated with 2ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, cell pellet, no staining on (C) untreated CXCL8 knockout PC-3 cell pellet and (D) CXCL8 knockout PC-3 treated with 2ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, cell pellet. The section was incubated with ab322732 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 Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL-8 antibody [RM1267] (ab322732)

  Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling IL-8 with ab322732 at 1/1000 (0.491 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  Positive staining on some immune cells in human colon carcinoma. The section was incubated with ab322732 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 Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

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  Immunocytochemistry/ Immunofluorescence - Anti-IL-8 antibody [RM1267] (ab322732)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CXCL8 KO PC-3 (CXCL8 knockout human prostate adenocarcinoma epithelial cell), Human CXCL8 knockout PC-3 cell line ab273743 cells labelling IL-8 with ab322732 at 1/2000 (0.246 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

  Confocal image showing increased cytoplasmic staining in wildype PC-3 cells treated with Lipopolysaccharide (2 ug/ml) and Brefeldin A (5 ug/ml) for 5 hours (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

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  Western blot - Anti-IL-8 antibody [RM1267] (ab322732)

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

  In Western blot, ab322732 and Anti-IL-8 antibody ab106350 were shown to bind specifically to IL-8. Target of interest was observed at 11 kDa in untreated wild-type PC-3 cell lysates (lane 1) and upregulated in treated wild-type PC-3 cell lysates (lane 2). No signal was observed at this size in untreated IL-8 knockout cell line (lane 3) or treated IL-8 knockout cell line (lane 4).

  Recombinant multiclonal ab322732 showed higher sensitivity than polyclonal Anti-IL-8 antibody ab106350.

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

  All lanes: Western blot - Anti-IL-8 antibody [RM1267] (ab322732) at 1/1000 dilution

  Lane 1: Untreated Wild-type PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: Wild-type PC-3 treated with2 ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, whole cell lysate at 20 µg

  Lane 3: Untreated CXCL8 knockout PC-3 whole cell lysate at 20 µg

  Lane 4: CXCL8 knockout PC-3 treated with 2 ug/ml LPS for 1 hour, 5ug/ml Brefeldin A was then added for additional 5 hours, whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 11 kDa, 36 kDa

  Exposure time: 180s