Anti-IL-8 antibody [RM1267]

• IHC-P

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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 (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 (ab209101).

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

• Flow Cyt (Intra)

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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 (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, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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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), ab273743 cells labelling IL-8 with ab322732 at 1/2000 (0.246 ug/ml) dilution, followed by 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).

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 2ug/ml dilution.

• IHC-P

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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

• IHC-P

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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 (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 (ab209101).

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

• IP

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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)(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 (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) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 10s

• WB

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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 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 ab106350.

In Western blot, 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 11 kDa,36 kDa

false

Exposure time: 180s

• I-ELISA

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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