Anti il 8 antibody

Western blot analysis was performed as previously described [27 (link)]. The cell lysate with SDS sample buffer was separated by denaturing SDS-PAGE. Separated proteins were transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk for 1 hour and incubated with primary antibodies overnight, including anti-USP21 antibody (Santa Cruz, 1:1000), anti-IL-8 antibody (Abcam 1:1000). β-Actin was used a loading control and was detected with a mouse mAb (Sigma-Aldrich, 1:2000). The immunocomplexes were treated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime Biotech) and detected using BeyoECL PLUS Kit (Beyotime Biotech). Cultured carcinoma cells A-704 and 786-O treated with Control or USP21 siRNA were plated on a 100 mm plate. IL-8 concentration was measured using the IL-8 ELISA kit (R&D systems). All assays were preformed as to the manufactures specifications.

Neutrophils were harvested from healthy volunteers and purified by Ficoll-Paque gradient centrifugation and dextran sedimentation. After hypotonic lysis of residual red blood cells, the neutrophils were used for further studies. Neutrophils were counted and resuspended in 1640 medium at a density of 5 × 10⁵ in 50 μL. Fifty microliters of cell suspension and 50 μL of plasma were used in each assay. Following an incubation at 37 °C for 2 h, CXCR-1 and CXCR-2 expression on CD66b positive cells were examined.
For anti-IL-8 antibody blocking, plasma from ACLF patient was incubated with anti-IL-8 antibody (Abcam, Camdridge, UK) at a concentration of 0.1 μg/mL for 1 h at room temperature, and then incubated with neutrophils for an additional 2 h to evaluate CXCR-1 and CXCR-2 expression.

Immunohistochemical staining was performed according to the manufacturer’s procedure with the Mouse and Rabbit Specific HRP/DAB IHC Detection Kit-Micro-polymer (Cat No. ab236466; Abcam, UK). Proteinase K (Cat No. ab64220; Abcam, UK) was used for antigen retrieval. Sections taken from paraffin blocks with adhesive were submerged in 3% H₂O₂ peroxidase block solution and then the protein block solution was poured out. The sections were exposed to 1:100 anti-TNF-α antibody (Cat No. ab6671; Abcam, USA), anti-IL-6 antibody (Cat No. ab6672; Abcam, USA), and anti-IL-8 antibody (Cat No. ab34100; Abcam, USA) and were left at room temperature for 1 h. Subsequently, Mouse Identification Reagent (Complementary) solution was added to the slides and they were exposed to goat anti-rabbit HRP-conjugate. Slides were stained with DAB (3,3′-diaminobenzidine tetrahydrochloride). After counterstaining with Mayer’s hematoxylin, slides were sealed with coverslips and evaluated under a light microscope (Leica DM 400B, Leica, Germany). Negative control slides were also stained according to the same procedure and PBS was used instead of primary antibodies. Immunohistochemical staining was scored semiquantitatively as revealing low (+), moderate (++), or high (+++) expression.