Immunohistochemical analysis was performed using previously established method33 (link). Following primary antibodies were used: anti-VCAM-1, anti-ANGPTL1 and anti-PLAT (Abcam, UK). As secondary antibody anti-rabbit/mouse ImPRESS universal staining kit (Vector Laboratories, CA, USA) was applied. Sites of reactions were visualized as previously described34 (link). Intensity was calculated according to 3 points scoring system (0, no staining; 1, weak; 2, mild; 3, strong staining).
Anti vcam 1
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-VCAM-1 is a lab equipment product that can be used to detect and measure the levels of VCAM-1 (Vascular Cell Adhesion Molecule-1) in various samples. VCAM-1 is a cell adhesion molecule that plays a role in the inflammatory response. The product provides a tool for researchers to study VCAM-1 expression and its relationship to different biological processes and disease states.
Automatically generated - may contain errors
Lab products found in correlation
Anti icam 1, by Abcam (13 mentions)
Anti β actin, by Merck Group (6 mentions)
Anti vimentin, by Cell Signaling Technology (3 mentions)
Anti bax, by Abcam (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti snail, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti cyclin b1, by Abcam (2 mentions)
Anti txnrd1, by Abcam (2 mentions)
Anti cleaved parp, by Abcam (2 mentions)
Anti cleaved caspase 3, by Abcam (2 mentions)
Ripa buffer, by Solarbio (2 mentions)
Anti β actin, by Abcam (2 mentions)
Secondary detection antibody, by Abcam (2 mentions)
Ipvh00010, by Merck Group (2 mentions)
Total nitric oxide assay kit, by Beyotime (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti iκbα, by Cell Signaling Technology (2 mentions)
33 protocols using anti vcam 1
1
Immunohistochemical Analysis of VCAM-1, ANGPTL1, and PLAT
2
Immunohistochemical Analysis of Vascular Markers
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For immunohistochemistry,21 (link) 4-μm thick serial sections were deparaffinized, rehydrated and, after antigen retrieval and nonspecific peroxidase blocking, incubated with mouse monoclonal anti-ICAM-1 (Pierce, IL, USA), anti-eNOS (Pierce), anti-Ki67 (Ventana Medical Systems, AZ, USA), anti-BrdU (YLEM, Avezzano, Italy), anti-human CD31 (Ventana), anti-CD4 (Ventana), and rabbit polyclonal anti-VCAM-1 (Abcam, CB, UK), anti-iNOS (Pierce), and anti-PlGF (Abcam). For rat tissues, mouse monoclonal anti-rat CD31 (BD Pharmingen, NJ, USA), was used. Morphometric evaluation of immunoreactivity was performed according to defined criteria (see Supplementary Materials and Methods ).
3
Targeting BRAF, AKT, and MEK in Cancer Cells
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The BRAF inhibitor vemurafenib (PLX4032), AKT inhibitor MK2206, and MEK inhibitor U0126 were all obtained from Selleck Chemicals (Houston, TX, USA). The reactive oxygen species (ROS) inhibitor NAC (N-acetyl-l -cysteine) was purchased from Beyotime (Shanghai, China). PLX4032 and U0126 were both dissolved in dimethylsulfoxide (DMSO) in 50 mM stock. MK22062 was dissolved in DMSO in 20 mM stock. NAC was dissolved in water in 50 mM stock. Primary antibodies were used as follows: anti-VCAM-1 was obtained from Abcam (Cambridge, UK), anti-ERK, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-AKT, anti-phospho-AKT (Ser473), anti-mTOR, anti-phospho-mammalian target of rapamycin (mTOR), anti-cleaved caspase-3, anti-cleaved poly (ADP-ribose) polymerase (PARP), anti-Bim, anti-Bcl-xl, anti-Mcl-1, anti-Vimentin, anti-Snail, anti-ATP-binding cassette sub-family G member 2 (ABCG2), anti-CD44, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies were all purchased from Cell Signaling Technology (Beverly, MA, USA).
4
Protein Expression Analysis of Cell Signaling Pathways
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After treatment, cells were washed 3× with ice-cold Hank's Balanced Salt Solution (HBSS) and lysed with Radioimmunoprecipitation Assay buffer (RIPA) buffer containing protease inhibitors (Boston Bioproducts BP-421) Son et al., 2013 (link). The protein content of each sample was determined by Pierce BCA protein assay. Aliquots of cell lysate were resolved on 10% to 12% sodium dodecyl sulfate–polyacrylamide gels and subsequently transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with the following primary antibodies: anti-KLK10 (BiossUSA bs-2531R, 1:1000), anti-GAPDH (Abcam ab23565, 1:2000), anti-β-actin (Sigma-Aldrich A5316, 1:2000), anti-VCAM1 (Abcam ab134047, 1:1000), anti-ICAM1 (Abcam ab53013, 1:1000), and anti-phospho-NFκB p65 S356 (Cell Signaling #3033, 1:1000) overnight at 4°C in 5% milk in TBST at the concentration recommended by the manufacturer, followed by secondary antibody addition for 1 hr at RT in 5% milk in TBST. Protein expression was detected by a chemiluminescence method (Son et al., 2013 (link)).
5
Endothelial Cell Surface Protein Expression
6
Comprehensive Antibody Panel for Fibrosis Analysis
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The following antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA): goat anti-COL1A1, anti-p-p70s6k (Thr389), and anti-Akt; mouse anti-α-SMA, anti-LC-3, anti-mTOR, and anti-GNMT; rat anti-CD3; and rabbit anti-p70s6k, anti-p62, anti-p-mTOR (Ser2448), and anti-COL4A2. Mouse anti-p-Akt (Ser473) and rabbit ant-iNOS and anti-MPO antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Rat anti-F4/80 and rabbit anti-ICAM-1 and anti-VCAM-1 antibodies were obtained from Abcam (Cambridge, MA, USA). Both the mouse anti-GAPDH antibody and Masson’s trichrome staining kit were obtained from Sigma–Aldrich (St. Louis, MO, USA). The mouse anti-TGF-β antibody was obtained from R&D Systems (Minneapolis, MN, USA).
7
Protein Expression Analysis in Esophageal Cancer Cells
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The total protein in transfected KYSE450 and KYSE510 cells was extracted and quantified with the RIPA buffer (Solarbio, China), which was supplemented with 1% proteinase inhibitor cocktail. The protein concentration was obtained with the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA). Next, the quantified protein was loaded into 10% or 12% SDS-PAGE for electrophoresis separation. The separated protein bands were then electronically transferred to the membranes (Sigma-Aldrich, USA). Following that, the membranes were put under ice for 2 h with 5% BSA at room temperature. This step was followed by the incubation of the membranes overnight with primary antibodies at 4 °C and 1.5 h incubation with secondary detection antibody (Cat# ab205718, Abcam, USA) at room temperature. Finally, the protein immunoblots were visualized using the Enhanced Chemiluminescent (ECL) Reagent Kit (Thermo Fisher Scientific. USA). The density of the blots was obtained using ImageJ software. All the primary antibodies were purchased from Abcam (USA), including anti-CyclinB1 (Cat# ab32053), anti-ICAM1 (Cat# ab53013), anti-VCAM1 (Cat# ab134047), anti-Cleaved PARP (Cat# ab32561), anti-Bax (Cat# ab32503), anti-Cleaved Caspase-3 (Cat# ab2302), anti-TXNRD1 (Cat# ab124954) and anti-β-actin (Cat# ab8227).
8
Protein Expression Analysis in Cells
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The total protein extracts were isolated using lysis buffer containing phosphatase and proteases. After protein content determination, proteins were blotted onto nitrocellulose membranes22 and incubated with anti-NADPH-oxidase 4 (Nox4, Abcam), anti-ICAM-1 (Pierce), anti-VCAM-1 (Abcam), anti-iNOS (Pierce), anti-eNOS (Pierce), and anti-α tubulin (Sigma Aldrich) antibodies. Specific complexes were quantified as reported.22
9
Western Blot Quantification of ICAM-1 and VCAM-1
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Western blot procedures were performed as described previously [20 ]. The protein concentration for homogenized liver lysates was evaluated with DC Protein Assay Kit (Bio Rad, Hercules, CA, USA). The protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After then, the protein was transferred to a PVDF membrane (IPVH00010; Millipore, Billerica, MA, USA). 5% skim milk diluted in tris-buffered saline with 0.1% Tween-20 (TBS-T) was used as a blocking reagent. For primary antibodies, anti-ICAM-1 (sc-7891; Santa Cruz Biotechnology), anti-VCAM-1 (ab134047; Abcam), and anti-β-actin (A1978; Sigma, St. Louis, MO, USA; and sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. After then, the membrane was rinsed three times in TBS-T and incubated with secondary antibody including goat-anti-rabbit (401393; Calbiochem, San Diego, CA, USA) or goat-anti-mouse (401253; Calbiochem) antibody. The membranes were developed with ECL solution (Translab, Seoul, Korea) and were exposed to Amersham™ Imager 680 (GE Healthcare, Uppsala, Sweden) or ImageQuant LAS 500 imager (GE Healthcare). For the internal standard, β-actin (Sigma) was used.
10
AEE Crystal Extraction and Assay
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Transparent AEE crystals with a purity of 99.5% according to RE-HPLC were prepared in the Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS. The H2O2 solution (cat number: 323381), dimethyl sulfoxide (DMSO) and Trypsin-EDTA were supplied by Sigma (St. Louis, MO, USA). Deionized water (18.25 MΩ) was prepared with a Direct-Q®3 system (Millipore, Bedford, MA, USA). The Annexin V/PE apoptosis detection kit was purchased from BD Biosciences (San Jose, CA, USA). Anti-ICAM-1, anti-VCAM-1, and anti-E-selectin antibodies were obtained from Abcam (Cambridge, MA, USA). The rat-soluble vascular cell adhesion molecule 1(sVCAM-1) ELISA kit, rat soluble intercellular adhesion molecule 1 (sICAM-1) ELISA kit, and rat soluble E-selectin (sE-selectin) ELISA kit were obtained from Elabscience (Wuhan, China); the advanced glycation end product (AGE) ELISA kit was purchased from Wuhan USCN Business Co., Ltd. (Wuhan, China). MS-grade acetonitrile was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Carboxymethylcellulose sodium (CMC-Na) was supplied by Tianjin Chemical Reagent Company (Tianjin, China)
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