500,000 cells were seeded in 10 cm dishes overnight. Cells were detached by 3 mM EDTA, washed in DMEM/10% FBS and fixed in 10% neutral formalin buffer. Cells were permeabilized in Triton X-100 at 37°C for 10 minutes, and incubated at a 1:100 dilution of anti-CXCR2 (Abcam cat no.14935) for 1 hour. Cells were washed in PBS 3 times and incubated with goat anti-Rabbit-Alexa-Fluor-488 (Invitrogen cat no.A11008) for 1 hour. Samples were read using an LSRII Flow Cytometer and normalized to unstained and secondary antibody only controls.
Anti cxcr2
Manufactured by Abcam
Sourced in United States
Anti-CXCR2 is a laboratory research tool that can be used to detect and quantify the CXCR2 protein. CXCR2 is a G-protein coupled receptor that plays a role in inflammatory and immune responses.
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Lab products found in correlation
Anti cxcl1, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Gastrin, by Santa Cruz Biotechnology (1 mentions)
Anti p63, by Santa Cruz Biotechnology (1 mentions)
Anti loricrin, by Abcam (1 mentions)
Anti gfp, by Abcam (1 mentions)
Enhanced chemiluminescence solution, by PerkinElmer (1 mentions)
Urea assay kit, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Alt assay kit, by Abcam (1 mentions)
Anti sars cov 2 nucleocapsid protein, by Sino Biological (1 mentions)
Ast activity assay kit, by Abcam (1 mentions)
Anti il 1β, by Cell Signaling Technology (1 mentions)
Anti cd206, by Abcam (1 mentions)
Anti cd8, by Abcam (1 mentions)
Opal 7 colour manual ihc kit, by PerkinElmer (1 mentions)
Vectra polaristm automated quantitative pathology imaging system, by Akoya Biosciences (1 mentions)
10 protocols using anti cxcr2
1
CXCR2 Expression Quantification
2
Comprehensive IHC Staining Protocol
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For IHC, tissues were harvested, fixed in 10% neutralized Formalin for 2 days and then stored in 70% ethanol until further processing. H&E staining, PAS staining and IHC on paraffin-sections were performed following standard protocols. The following antibodies were used for IHC: anti-GFP (Abcam, 6673, 1:200; Clontech, JL-8, 1:100); Ki67 (Cell signaling, 12202, 1:200); Proton-pump (MBL, D032-3H, 1:100); Gastrin (Santa Cruz, sc-783, 1:200); anti-p63 (Santa Cruz, sc-56188, 1:200); anti-CK13 (Abcam, 92551, 1:1000); anti-Loricrin (Abcam, 24722, 1:1000); anti-CXCL7 (Bioss Inc., A-21235, 1:200); anti-CXCR2 (Abcam, 14935, 1:200).
3
Evaluating Organ Damage Biomarkers
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Multiple-organ damage was evaluated by determining the levels of serum AST and ALT (markers of liver injury) and BUN (a marker of kidney injury). AST, ALT, and BUN levels were measured using an AST activity assay kit (no. ab105135), ALT assay kit (no. ab105134), and urea assay kit (no. ab83362) (Abcam), respectively, according to the manufacturer’s instructions. For Western blot analysis, cellular or viral proteins were detected in lung homogenates using anti-SARS-CoV-2 nucleocapsid protein (no. 40143-R001; Sino Biological), anti-β-actin (no. sc-47778; Santa Cruz Biotechnology), anti-CXCR2 (no. ab217314; Abcam), and anti-IL-1β (no. 12242; Cell Signaling Technology) antibodies. Blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). Membranes were developed with enhanced chemiluminescence solution (PerkinElmer) and imaged using an ImageQuant Las 4000 system (GE Healthcare). Protein band intensities were quantified using ImageJ software (National Institutes of Health) (68 (link)). Data are presented as means ± standard deviations from three independent experiments.
4
Multiplex Immunohistochemistry of Pancreatic Cancer
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Fluorescent mIHC was performed in serial sections of FFPE tumor tissue from each PDAC patient using the Opal 7-Colour Manual IHC Kit (PerkinElmer Hopkinton, Massachusetts, USA) according to the manufacturer’s protocol and as described in our previous article. In short, sections experienced primary antibodies including Rabbit monoclonal antibodies anti-CD68 (1:500, Abcam, Cambridge, MA, USA), anti-CXCR2 (1:500, Abcam), anti-CD206 (1000, Abcam), anti-CXCL1 (1:200, Abcam), anti-CXCL5 (1:200, Abcam), anti-CXCL8 (1:500, Abcam) and anti-CD8 (1:2000, Abcam), followed by HRP-conjugated secondary antibody and fluorescent dyes (Opal520, Opal570, Opal620, Opal690, and DAPI). Stained sections were scanned and processed by a Vectra PolarisTM Automated Quantitative Pathology Imaging System (AKOYA Biosciences, PerkinElmer, Massachusetts, USA).
5
Immunofluorescence Assay for BrdU and CXCR2
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Cells were fixed on glass slides with methanol 20%(v/v), embedded in HCL 2M for 30 minutes at RT, incubated with anti-BrdU (1:10) or anti-CXCR2 (1:200) (Abcam) for 60 minutes at RT, then incubated with Alexa Fluor 494 goat anti-mouse or Alexa Fluor 494 goat anti-rabbit secondary antibodies (1:100) (Invitrogen) for 60 minutes, at RT, in the dark. Cells nuclei were stained with DAPI (1:10) (Thermo Fisher) for 15 minutes at RT. Images were acquired in Zeiss Z1 apotome microscope (LEICA) at 10x magnification.
6
Western Blot Analysis of CXCR1 and CXCR2 in Liver Cancer Cells
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In brief, liver cancer cells were lysed using radioimmunoprecipitation assay buffer (BIOSS) for 30 min on ice. The cell lysate was centrifuged at 12,000 × g for 20 min at 4°C and the supernatant was collected. Protein concentration was determined by BCA protein assay. A total of 30 µg protein/lane was separated by SDS-PAGE on a 10% gel and transferred onto nitrocellulose filter membranes. Following blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C, and then with secondary antibodies for 2 h at room temperature. The primary antibodies used were as follows: Rabbit polyclonal anti-CXCR1 (1:2,000; cat. no. ab137351; Abcam) and anti-CXCR2 (1:2,000; cat. no. ab14935; Abcam) antibodies; monoclonal mouse anti-GAPDH (1:2,000; cat. no. TA-08; Zhongshan Jinqiao Biotechnology) and β-actin (1:2,000, cat. no. TA-09; Zhongshan Jinqiao Biotechnology, Beijing, China). The goat anti-mouse (cat. no. ZB2305; 1:10,000) and anti-rabbit secondary antibodies (cat. no. ZB2301; 1:10,000) were purchased from Zhongshan Jinqiao Biotechnology. The protein bands were visualized by ECL reagents (cat. no. C05-07003; BIOSS). Images were captured and the intensity of the bands was quantitated with the Bio-Rad Versa Doc imaging system (Bio-Rad Laboratories, Inc.).
7
Western Blot Analysis of CXCL1, CXCR2, and NF-κB
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The RIPA buffer was performed to prepare the total protein lysates from SH-SY5Y cells or rat tissues, and the total protein was quantified with the BCA protein assay kit (Beyotime, Shanghai, China). Then, protein samples were separated on 10% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skimmed milk in TBST for 1 h. Then, the membranes were incubated with primary antibodies, namely, anti-CXCL1 (Abcam, Cambridge, MA, USA; 1:1000), anti-CXCR2 (Abcam; 1:1000), anti-p65 (Abcam; 1:1000), anti-p-p65 (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000) at 4°C overnight. Subsequently, the membranes were incubated with the secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. The membranes were then developed with ECL, scanned using an Odyssey Imaging System, and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, USA) to determine protein band intensity. The levels of CXCL1, CXCR2, p65, and phospho-p65 were measured relative to β-actin, which was used as an internal loading control.
8
Effect of IL-8 on Liver Cancer Migration
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The Huh-7 and HepG2 human liver cancer cell lines were purchased from the Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. The HepG2 cell line used in the present study has been authenticated by STR profiling. The human Huh-7 and HepG2 cell lines were cultured in DMEM (Hyclone; GEHealthcare Life Sciences) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2. In order to observe the effect of IL-8 on the migration and invasion of liver cancer cell lines, the Huh-7 and HepG2 cells (1.0×106) were incubated with IL-8 at 37°C for 24 and 48 h. In certain cases, the Huh-7 and HepG2 cells were pretreated with 5 µM anti-CXCR1 (1:400, cat. no. ab137351; Abcam) or anti-CXCR2(1:400; cat. no. ab14935; Abcam) for 30 min at 37°C to inhibit the function of CXCR1 or CXCR2, and the migration and invasion were measured in vitro via a wound healing assay and Transwell assay.
9
Immunocytochemical Analysis of IL-8, CXCR1, and CXCR2
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The SKOV3 and A2780 cells were digested by trypsin‐EDTA and seeded into 24 cell plates. Then, the cells were incubated at 37°C, washed with PBS and fixed in 4.00% paraformaldehyde for 15 minutes. After permeablization using 0.50% Triton X‐100 and blocking by 5.00% normal goat serum, the cells were incubated with the using of anti–IL‐8, anti‐CXCR1 and anti‐CXCR2 (1:100, Abcam) antibodies at 4°C overnight, and the second antibody was incubated for 1 hours at room temperature. Finally, the cells were counterstained with haematoxylin and visualized with the DAB.
10
Western Blot Analysis of Cell Signaling Proteins
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Protein preparations from the indicated cells were obtained by lysing samples in 50 mmol/L of TRIS (pH 7.5), 100 mmol/L of NaCl, 1% NP40, 0.1% Triton, 2 mmol/L of EDTA, 10 Ag/mL of aprotinin, and 100 Ag/mL of phenylmethylsulfonyl-fluoride. Prestained molecular weight standards were from Bio-Rad (Milan). Proteins separated on the polyacrylamide gels were blotted on a PVDF membrane. The membrane was stained with Ponceau S (Sigma) to enable us to evaluate the success of transfer, and to locate the molecular weight markers. Free protein binding sites on the PVDF membrane were blocked with nonfat dry milk and a Tween 20/TBS solution. The membranes were washed and stained with specific primary antibodies and with secondary antisera, and then conjugated with horseradish peroxidase (Sigma-Aldrich) diluted 1:2,000. The anti-E-cadherin, anti-N-cadherin, anti-CXCR2, anti-phospho-P38, anti-P38, anti-IκB, anti-NF-κB, anti-slug, anti-twist1, anti-ZEB1, anti-Snail and anti-SIP1 antibodies were purchased from Abcam, and the anti-GRO-α antibody was from Santa Cruz. The luminescent signal was visualized with the ECL Western blotting detection reagent kit (Amersham) and quantified by scanning with a Discover Pharmacia scanner equipped with a Sun Spark Classic Workstation.
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