IHC was performed according to standard protocols. Expression levels of IL8 (Abcam, Cambridge, UK, 1:1000) and CXCR7 (Abcam, Cambridge, UK, 1:800) were detected using IHC. All sections were assessed at 20× magnification by one pathologist and two experienced observers, and visualized under a microscope (Olympus, Japan). Staining was evaluated based on intensity (negative = 0; weak = 1; moderate = 2; and high = 3) of immunostaining and density (0% = 0; 1–40% = 1; 41–75% = 2; > 76% = 3) of positive tumor cells. The final immunoreactivity score of each sample was acquired by multiplying the intensity and density scores.
Cxcr7
Manufactured by Abcam
Sourced in United Kingdom, United States
CXCR7 is a chemokine receptor that binds to the chemokines CXCL11 and CXCL12. It is involved in various cellular processes, including cell migration and signaling.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
Cxcr4, by R&D Systems (2 mentions)
Vascular endothelial growth factor (vegf), by Abcam (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Sdf 1, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence kit, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Snail, by Abcam (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
E cadherin, by Abcam (1 mentions)
Mmp 9, by Abnova (1 mentions)
Hepatocyte growth factor (hgf), by Abcam (1 mentions)
Smad4, by Abcam (1 mentions)
Masson trichrome, by Abcam (1 mentions)
Sdf 1α, by Abcam (1 mentions)
C met, by Abcam (1 mentions)
9 protocols using cxcr7
1
IHC Evaluation of IL8 and CXCR7
2
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted with a protein kit (Solarbio). The protein samples were denaturalized in boiling water. Equal amount of protein samples was loaded on each lane and separated on SDS‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (EMD Millipore). Afterwards, the membranes were blocked with 5% non‐fat milk and incubated with primary antibodies against SDF‐1 (Abcam) and CXCR7 (Abcam) over night at 4°C. Then, the membranes were rinsed with Tris‐buffered saline Tween‐20 (TBST) thrice, incubated with a secondary horseradish peroxidase‐conjugated antibody (Cell Signaling) for 2 hours at room temperature. Immunoblot signals were detected by an enhanced chemiluminescence system (ECL kit) and quantified by scanning densitometry using the ImageJ analysis system (NIH).
3
Immunoblot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from fresh tissues and cells, separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA), and subjected to immunoblot analyses. Blotting was performed with primary antibodies targeting CXCR7, E-cadherin, Snail (all from Abcam, Cambridge, UK), cyclin D1, MMP2, MMP9 (all from Abnova, Taiwan, China), STAT3, p-STAT3 (Tyr-705), Bcl-2, Mcl-1 (all from Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma). The signals were detected using a horseradish-peroxidase-conjugated secondary antibody (Sigma), and the bands were visualized using an enhanced chemiluminescence kit (Santa Cruz, Dallas, TX, USA). Protein bands were quantified using Quantity One software (BioRad, USA).
4
Comprehensive Immunohistochemical Analysis of Tumor Microenvironment
5
Molecular Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The transfected cells were lysed in 100 μL of ice‐cold RIPA buffer (Beyotime, Shanghai, China) for 15 minutes and collected by scraping into individual Eppendorf tubes. The antibodies and their dilutions were as follows: CXCR7 (1:1000), VEGF (1:2000), ERK (1:10000), p‐ERK 1:5000, GAPDH (1:2000) (Abcam, Cambridge, UK), and AKT (1:1000), and p‐AKT Thr308 (1:1000) (Cell Signaling Technology, Danvers, Mass). The protein bands were detected using an Image Lab software and displayed using photographic film.
6
Phenotypic Characterization of GMP-ASC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GMP‐ASC alone and treated either with OA‐CM or OA‐SF were characterized by flow cytometry using the following markers CXCR1, CXCR3, CXCR4, CCR1, CCR2, CCR3, CCR5 (R&D), CXCR7 (Abcam, Cambridge, UK), and IL6R (GeneTex Inc., Irvine, CA).
In brief, after harvesting cells upon detachment, they were washed twice with phosphate‐buffered saline (PBS), centrifuged, and washed in a flow cytometry buffer (PBS supplemented with 2% bovine serum albumin [BSA] and 0.1% sodium azide).
Aliquots of 1 × 105 cells were then incubated with primary antibodies at 10 µg/ml 4 °C for 30 min, washed twice with a flow cytometry buffer, and incubated with polyclonal rabbit anti‐mouse and goat anti‐rabbit immunoglobulins/fluorescein isothiocyanate (FITC) conjugate (Dako Cytomation, Glostrup, Denmark) at 4 °C for 30 min. After two final washes, the cells were analyzed using a fluorescence‐activated cell sorting (FACS) CantoII Cytometer (Becton Dickinson). For isotype control, non‐specific mouse IgG was substituted for the primary antibody.
In brief, after harvesting cells upon detachment, they were washed twice with phosphate‐buffered saline (PBS), centrifuged, and washed in a flow cytometry buffer (PBS supplemented with 2% bovine serum albumin [BSA] and 0.1% sodium azide).
Aliquots of 1 × 105 cells were then incubated with primary antibodies at 10 µg/ml 4 °C for 30 min, washed twice with a flow cytometry buffer, and incubated with polyclonal rabbit anti‐mouse and goat anti‐rabbit immunoglobulins/fluorescein isothiocyanate (FITC) conjugate (Dako Cytomation, Glostrup, Denmark) at 4 °C for 30 min. After two final washes, the cells were analyzed using a fluorescence‐activated cell sorting (FACS) CantoII Cytometer (Becton Dickinson). For isotype control, non‐specific mouse IgG was substituted for the primary antibody.
7
Protein Extraction and Western Blot Analysis of MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis buffer supplemented with 1 mmol/l phenylmethanesulfonyl fluoride (Beyotime) were used to extract total proteins of MSCs. Extract total proteins of MSCs were separated with SDS-PAGE condensed electrophoresis (Beyotime) and transferred onto polyvinylidene fluoride membranes (Beyotime). Then, we blocked the membranes in 5% BSA and dyed them for 1 h at room temperature and dyed at 4 °C overnight with primary antibodies against β-Actin (1:1000; Cell Signaling, USA), HIF-1α (1:1000;Cell Signaling), Hyp564 HIF-1α (1:1000;Cell Signaling), CXCR4 (1:100; Abcam, USA), CXCR7(1:1000;Abcam), Caspase-3 (1:1000;Cell Signaling), and Cleaved-Caspase-3 (1:1000;Cell Signaling). Peroxidase-conjugated secondary antibody (1:3000; Fcmacs) was used to incubate the membrane at room temperature for 1 h. Finally, ECL was applied to detect the bands with a chemiluminescence imaging system (Bioshine ChemiQ 4800mini, Ouxiang, Shanghai, China).
8
Protein Expression Analysis in Exosomes and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins from exosomes, EPCs, ADSCs and mouse myocardial tissues were extracted by using lysates. The bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology, Jiangsu, China) was used to determine the protein concentration of each sample. Forty micrograms of total protein was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Jaffrey, NH, USA). After blocking in 5% nonfat milk for 1 h, the membranes were incubated with primary antibodies against CXCR7 (1:500; Abcam), SIRT1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD63(1:500; Abcam), TSG101(1:500; Abcam), Nrf2 (1:500; Abcam), CXCL12 (1:1,000; Abcam), or β-actin (1:2,000; Abcam) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. The signals were detected using an enhanced chemiluminescence (ECL) detection system (Thermo Scientific, Rockford, IL, USA). The relative protein levels were determined after normalization with β-actin. Densitometric analysis of the bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
9
Immunohistochemical Analysis of Tumor Angiogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sections of tumors were prepared for immune histochemical analysis, embedded in paraffin, and cut into slices. After dewaxing, dehydrating, repairing, inactivating, and blocking, the tissue sections were incubated with CD34 (1:2500), CXCR7 (1:200) antibody, or VEGF (1:100) antibodies (Abcam), overnight at 4°C. After incubation with the secondary antibody, the sections were stained using diaminobenzidine. Cytoplasmic staining of vascular endothelial cells was considered positive.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!